Compositions and methods for the modulation of dna damage responses using bal1 and bbap

ABSTRACT

The invention provides methods and compositions for enhancing the efficacy of cancer therapies through modulation of BAL1 and/or BBAP. Also provided are methods for predicting the efficacy of cancer therapies or treating cancer in a subject through modulation of BAL1 and/or BBAP. Further provided are methods for identifying compounds that are capable of modulating BAL1-BBAP complexes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/543,465, filed on Oct. 5, 2011; the entire content of saidapplication is incorporated herein in its entirety by this reference.

STATEMENT OF RIGHTS

This invention was made with government support under grant numberPO1CA092625 awarded by The National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND OF THE INVENTION

Cancer is a global health concern affecting millions of people. Since itrepresents the phenotypic end-point of multiple genetic lesions, cancerendows cells with a full range of biological properties required fortumorigenesis. This complex myriad of biological causes resulting in thesame phenotype indicates that novel compositions and methods foreffectively diagnosing and treating cancer are needed to combat theineffectiveness of any given cancer therapy. For example,chemotherapeutics can work by interfering with cell cycle progression orby generating DNA strand breaks. If the cancer cell is not able toovercome the cell cycle blockage or cell injury caused by thetherapeutic compound, the cell will often die via apoptotic mechanisms.Yet cancer cells commonly develop resistance to the chemotherapeuticagent thereby rendering a given chemotherapeutic agent or cancer therapytargeting a certain biological mechanism ineffective. Despite decades ofscientific research, few effective therapies have emerged to targetalternative biological pathways critical to the development of cancer.Accordingly, there is a great need to identify compositions and methodsto effectively target such biological pathways.

SUMMARY OF THE INVENTION

The present invention provides methods and compositions for enhancingthe efficacy of cancer therapies through modulation of BAL1 and/or BBAP.In one aspect, a method of treating cancer by enhancing the efficacy ofcancer therapies in a subject, comprising administering to the subjectan effective amount of (a) an agent that inhibits one or more functionsof BAL1, BBAP, or a BAL1-BBAP complex and (b) the cancer therapy isprovided. In one embodiment, the cancer is selected from the groupconsisting of breast cancer, ovarian cancer, transitional cell bladdercancer, bronchogenic lung cancer, thyroid cancer, pancreatic cancer,prostate cancer, uterine cancer, testicular cancer, gastric cancer, softtissue and osteogenic sarcomas, neuroblastoma, Wilms' tumor, malignantlymphoma (Hodgkin's and non-Hodgkin's), acute myeloblastic leukemia,acute lymphoblastic leukemia, Kaposi's sarcoma, Ewing's tumor,refractory multiple myeloma, and squamous cell carcinomas of the head,neck, cervix, colon cancer, melanoma, and vagina. In another embodiment,the cancer therapy is selected from the group consisting ofchemotherapy, radiation therapy, immunotherapy, hormone therapy,hyperthermic, laser therapy, gene therapy, PARP inhibitor therapy, andcombinations thereof (e.g., treatment with a PARP-1 inhibitor). In stillanother embodiment, the agent is selected from the group consisting ofan antibody or an antigen binding fragment thereof, which specificallybinds to a protein corresponding to BAL1, BBAP, and/or a BAL1-BBAPcomplex; an RNA interfering agent which inhibits expression of BAL1and/or BBAP; an antisense oligonucleotide complementary to BAL1 and/orBBAP; a small molecule which inhibits activity of BAL1, BBAP, and/or aBAL1-BBAP complex; an aptamer which inhibits expression or activity ofBAL1, BBAP, and/or a BAL1-BBAP complex; and a BAL1 and/or BBAPpolypeptide described herein. In such embodiments, the antibody can beconjugated to a toxin or a chemotherapeutic agent; or the RNAinterfering agent is an siRNA molecule or an shRNA molecule; or thesmall molecule inhibits a protein-protein interaction between BAL1 andBBAP. In yet another embodiment, the efficacy of treatment is measuredby at least one criteria selected from the group consisting of survivaluntil mortality, pathological complete response, semi-quantitativemeasures of pathologic response, clinical complete remission, clinicalpartial remission, clinical stable disease, recurrence-free survival,metastasis free survival, disease free survival, circulating tumor celldecrease, circulating marker response, and RECIST criteria. In anotherembodiment, the subject is a human.

In another aspect, a method of enhancing the efficacy of cancertherapies in inhibiting hyperproliferation of a hyperproliferative cellin a medium, comprising applying to said medium an effective amount of(a) an agent that inhibits one or more functions of BAL1, BBAP, or aBAL1-BBAP complex and (b) the cancer therapy is provided. In oneembodiment, the hyperproliferative cells are selected from the groupconsisting of breast cancer cells, ovarian cancer cells, transitionalcell bladder cancer cells, bronchogenic lung cancer cells, thyroidcancer cells, pancreatic cancer cells, prostate cancer cells, uterinecancer cells, testicular cancer cells, gastric cancer cells, soft tissueand osteogenic sarcoma cells, neuroblastoma cells, Wilms' tumor cells,malignant lymphoma (Hodgkin's and non-Hodgkin's) cells, acutemyeloblastic leukemia cells, acute lymphoblastic leukemia cells,Kaposi's sarcoma cells, Ewing's tumor cells, refractory multiple myelomacells, and squamous cell carcinoma of the head, neck, cervix, coloncancer, melanoma, and vagina cells. In another embodiment, cancertherapy is selected from the group consisting of chemotherapy, radiationtherapy, immunotherapy, hormone therapy, hyperthermic therapy, lasertherapy, gene therapy, PARP inhibitor therapy, and combinations thereof(e.g., treatment with a PARP-1 inhibitor). In still another embodiment,the agent is selected from the group consisting of an antibody or anantigen binding fragment thereof, which specifically binds to a proteincorresponding to BAL1, BBAP, and/or a BAL1-BBAP complex; an RNAinterfering agent which inhibits expression of BAL1 and/or BBAP; anantisense oligonucleotide complementary to BAL1 and/or BBAP; a smallmolecule which inhibits activity of BAL1, BBAP, and/or a BAL1-BBAPcomplex; an aptamer which inhibits expression or activity of BAL1, BBAP,and/or a BAL1-BBAP complex; and a BAL1 and/or BBAP polypeptide describedherein. In such embodiments, the antibody can be conjugated to a toxinor a chemotherapeutic agent; or the RNA interfering agent is an siRNAmolecule or an shRNA molecule; or the small molecule inhibits aprotein-protein interaction between BAL1 and BBAP. In yet anotherembodiment, the efficacy of treatment is measured by at least onecriteria selected from the group consisting of survival until mortality,pathological complete response, semi-quantitative measures of pathologicresponse, clinical complete remission, clinical partial remission,clinical stable disease, recurrence-free survival, metastasis freesurvival, disease free survival, circulating tumor cell decrease,circulating marker response, and RECIST criteria. In another embodiment,the cells are human cells.

In still another aspect, a method of predicting the efficacy of a cancertherapy in a subject, comprising obtaining a biological sample from thesubject, and comparing: a) the amount, structure, subcellularlocalization, and/or activity of at least one marker selected from thegroup consisting of BAL1, BBAP, and/or BAL1-BBAP complex in a subjectsample; and b) the amount, structure, subcellular localization, and/oractivity of the at least one marker in a control, wherein a significantdifference in the amount, structure, subcellular localization, and/oractivity of the at least one marker in the sample and the amount,structure, subcellular localization, and/or activity in the control ispredictive of the outcome of treatment of the subject with the cancertherapy. In one embodiment, the control is selected from the groupconsisting of a non-cancerous cell sample from the subject or member ofthe same species to which the subject belongs; a non-cancerous tissuethat is the same tissue type as the cancerous tissue of the subject; anda non-cancerous tissue that is not the same tissue type as the canceroustissue of the subject. In another embodiment, the control amount,subcellular localization, structure, and/or activity is the wild typeamount, subcellular localization, structure, and/or activity in thespecies to which the subject belongs. In still another embodiment, thesubject sample is obtained before the subject has received cancertherapy or the subject sample is obtained after the subject has receivedcancer therapy. In yet another embodiment, the sample is selected fromthe group consisting of cells, cell lines, histological slides, paraffinembedded tissues, biopsies, whole blood, nipple aspirate, serum, plasma,buccal scrape, saliva, cerebrospinal fluid, urine, stool, and bonemarrow. In another embodiment, the cancer is selected from the groupconsisting of breast cancer, ovarian cancer, transitional cell bladdercancer, bronchogenic lung cancer, thyroid cancer, pancreatic cancer,prostate cancer, uterine cancer, testicular cancer, gastric cancer, softtissue and osteogenic sarcomas, neuroblastoma, Wilms' tumor, malignantlymphoma (Hodgkin's and non-Hodgkin's), acute myeloblastic leukemia,acute lymphoblastic leukemia, Kaposi's sarcoma, Ewing's tumor,refractory multiple myeloma, and squamous cell carcinomas of the head,neck, cervix, and vagina. In still another embodiment, the amount of themarker is determined by determining the level of expression or copynumber of the marker. In yet another embodiment, the copy number isdetermined by using at least one technique selected from the groupconsisting of fluorescence in situ hybridization (FISH), quantitativePCR (qPCR), comparative genomic hybridization (CGH), andsingle-nucleotide polymorphism (SNP) array. In another embodiment, anincreased copy number in the subject sample indicates reduced efficacyof the cancer therapy and a decreased copy number in the subject sampleindicates increased efficacy of the cancer therapy, thereby predictingthe efficacy of the cancer therapy in the subject. In still anotherembodiment, the level of expression of the marker in the sample isassessed by detecting the presence in the sample of a proteincorresponding to the marker (e.g., by using a reagent selected from thegroup consisting of an antibody, an antibody derivative, and an antibodyfragment). In yet another embodiment, the level of expression of themarker in the sample is assessed by detecting the presence in the sampleof a transcribed polynucleotide (e.g., mRNA or cDNA) or portion thereof,wherein the transcribed polynucleotide comprises the marker. In anotherembodiment, determining the level of expression of the marker comprisesthe use of at least one technique selected from the group consisting ofNorthern blot analysis, reverse transcriptase PCR, real-time PCR, RNAseprotection, and microarray analysis. In still another embodiment, thelevel of expression of the marker in the sample is assessed by detectingthe presence in the sample of a transcribed polynucleotide which annealswith the marker or anneals with a portion of a polynucleotide whereinthe polynucleotide comprises the marker, under stringent hybridizationconditions. In yet another embodiment, the significant difference is anincrease in the amount, structure, subcellular localization, and/oractivity of the marker in the subject sample relative to the control,indicating a reduced likelihood of efficacy of the cancer therapy in thesubject. In another embodiment, the significant difference is a decreasein the amount, structure, subcellular localization, and/or activity ofthe marker in the subject sample relative to the control, indicating anincreased likelihood of efficacy of the cancer therapy in the subject.In still another embodiment, the efficacy of treatment is measured by atleast one criteria selected from the group consisting of survival untilmortality, pathological complete response, clinical complete remission,clinical partial remission, clinical stable disease, recurrence-freesurvival, metastasis free survival, and disease free survival. In yetanother embodiment, cancer therapy is selected from the group consistingof chemotherapy, radiation therapy, immunotherapy, hormone therapy,hyperthermic therapy, laser therapy, gene therapy, PARP inhibitortherapy, and combinations thereof (e.g., treatment with a PARP-1inhibitor).

In yet another aspect, the present invention provides a use of an agentthat that inhibits one or more functions of BAL1, BBAP, or a BAL1-BBAPcomplex for the preparation of a medicament for enhancing the efficacyof a cancer therapy in a subject. In one embodiment, the one or morefunctions of BAL1, BBAP, or a BAL-BBAP complex is inhibited using anagent selected from the group consisting of an anti-BAL1 and/oranti-BBAP antisense nucleic acid molecule; an anti-BAL1 and/or anti-BBAPRNA interference molecule; a blocking anti-BAL1, anti-BBAP, and/oranti-BAL1-BBAP antibody; a non-activating form of BAL1, BBAP, or aBAL1-BBAP polypeptide or fragment thereof; and a small molecule thatbinds to BAL1, BBAP, or a BAL1-BBAP. In another embodiment, the usefurther comprises contacting the cell with an additional cancertherapeutic agent.

In another aspect, a method for assessing the efficacy of an agent thatmodulates the expression and/or activity of BAL1, BBAP, or a BAL-BBAPcomplex for enhancing the efficacy of a cancer therapy in a subjectcomprising a) detecting in a subject sample at a first point in time,the expression and/or activity of BAL1, BBAP, or a BAL-BBAP complex; b)repeating step a) during at least one subsequent point in time afteradministration of the agent; and c) comparing the expression and/oractivity detected in steps a) and b), wherein a significantly higherexpression and/or activity of BAL1, BBAP, or a BAL-BBAP complexexpression and/or activity in the first subject sample relative to atleast one subsequent subject sample, indicates that the agent enhancesthe efficacy of the cancer therapy in the subject and/or wherein asignificantly decreased amount of an activity selected from the groupconsisting of a) increased binding to a BAL1 polypeptide or fragmentthereof; b) increased binding of a BBAP polypeptide or fragment thereof;c) increased formation of a BAL1-BBAP complex; d) inhibition oflocalization and/or binding of BAL1 and/or BBAP to DNA damage sites; e)inhibition of binding of BAL1 to poly(ADP-ribose) (PAR) chains; f)inhibition of BBAP monoubiquitylation of histones; g) inhibition ofBBAP-mediated methylation of histones; h) inhibition of localizationand/or binding to DNA damage sites of at least one polypeptide selectedfrom the group consisting of 53 BP1, RAP80, BRCA1, ATM, γH2AX, and MDC1;and i) inhibition of DNA damage responses (DDR); in the first subjectsample relative to at least one subsequent subject sample, indicatesthat the test agent enhances the efficacy of the cancer therapy in thesubject is provided. In one embodiment, the agent is selected from thegroup consisting of an anti-BAL1 and/or anti-BBAP antisense nucleic acidmolecule; an anti-BAL1 and/or anti-BBAP RNA interference molecule; ablocking anti-BAL1, anti-BBAP, and/or anti-BAL1-BBAP antibody; anon-activating form of BAL1, BBAP, or a BAL1-BBAP polypeptide orfragment thereof; and a small molecule that binds to BAL1, BBAP, or aBAL1-BBAP. In another embodiment, the subject has undergone cancertherapy treatment, has completed cancer therapy treatment, and/or is inremission from the cancer between the first point in time and thesubsequent point in time. In still another embodiment, the first and/orat least one subsequent sample is selected from the group consisting ofex vivo and in vivo samples. In yet another embodiment, the first and/orat least one subsequent sample is obtained from an animal model of acancer. In another embodiment, the first and/or at least one subsequentsample is selected from the group consisting of tissue, whole blood,serum, plasma, buccal scrape, saliva, cerebrospinal fluid, urine, stool,and bone marrow. In still another embodiment, the first and/or at leastone subsequent sample is a portion of a single sample or pooled samplesobtained from the subject. In yet another embodiment, a significantlymodulated expression and/or activity comprises modulating the expressionand/or activity by at least 25% relative to the second sample.

In still another aspect, an isolated nucleic acid molecule selected fromthe group consisting of a) an isolated nucleic acid molecule whichencodes at least one BBAP binding domain of a BAL1 protein and whichdoes not encode full-length BAL1; b) an isolated nucleic acid moleculewhich encodes at least one BBAP binding domain of a BAL1 protein andwhich does not encode one or more functional domain(s) of a BAL1 proteinselected from the group consisting of Macro1, Macro 2, and PARP domains;c) an isolated nucleic acid molecule which encodes a polypeptidecomprising an amino acid sequence having at least 70% identity to theamino acid sequence of 453-702 of SEQ ID NO:2 and which does not encodefull-length BAL1 and/or which does not encode one or more functionaldomain(s) of a BAL1 protein selected from the group consisting ofMacro1, Macro 2, and PARP domains; d) an isolated nucleic acid moleculewhich encodes a polypeptide consisting essentially of an amino acidsequence having at least 70% identity to the amino acid sequence of453-702 of SEQ ID NO:2 and which does not encode full-length BAL1 and/orwhich does not encode one or more functional domain(s) of a BAL1 proteinselected from the group consisting of Macro1, Macro 2, and PARP domains;e) an isolated nucleic acid molecule which encodes at least one BAL1binding domain of a BBAP protein and which does not encode full-lengthBBAP; f) an isolated nucleic acid molecule which encodes at least oneBAL1 binding domain of a BBBAP protein and which does not encode one ormore functional domain(s) of a BBAP protein selected from the groupconsisting of BBAP dimerization and RING domains; g) an isolated nucleicacid molecule which encodes a polypeptide comprising an amino acidsequence having at least 70% identity to the amino acid sequence of423-617 of SEQ ID NO:10 and which does not encode full-length BBAPand/or which does not encode one or more functional domain(s) of a BBAPprotein selected from the group consisting of BBAP dimerization and RINGdomains; and h) an isolated nucleic acid molecule which encodes apolypeptide consisting essentially of an amino acid sequence having atleast 70% identity to the amino acid sequence of 423-617 of SEQ ID NO:10and which does not encode full-length BBAP and/or which does not encodeone or more functional domain(s) of a BBAP protein selected from thegroup consisting of BBAP dimerization and RING domains, as well asisolated nucleic acid molecules comprising a nucleotide sequence whichis complementary to such nucleic acid sequences, is provided. In oneembodiment, the isolated nucleic acid molecules further comprise anucleic acid sequence encoding a heterologous polypeptide (e.g., asignal peptide, a peptide tag, a dimerization domain, an oligomerizationdomain, an antibody, or an antibody fragment).

In yet another aspect, vectors (e.g., an expression vector) comprisingisolated nucleic acid molecules described herein are provided.

In another aspect, host cells transfected with a vector described hereinare provided.

In still another aspect, a method of producing a polypeptide comprisingculturing host cells described herein in an appropriate culture mediumto thereby produce the polypeptide are provided. In one embodiment, thehost cell is selected from the group consisting of a bacterial cell, aeukaryotic cell, and a cell genetically engineered to express aselectable marker. In another embodiment, the method further comprisesthe step of isolating the polypeptide from the medium or host cell.

In yet another aspect, an isolated polypeptide selected from the groupconsisting of a) an isolated polypeptide fragment of a BAL1 proteincomprising at least one BBAP binding domain and is not full-length BAL1;b) an isolated polypeptide fragment of a BAL1 protein comprising atleast one BBAP binding domain and which lacks one or more functionaldomain(s) selected from the group consisting of Macro1, Macro 2, andPARP domains; c) an isolated polypeptide comprising an amino acidsequence that is at least 70% identical to the amino acid sequence ofresidues 453-702 of SEQ ID NO:2 and is not full-length BAL1 and/or whichlacks one or more functional domain(s) of a BAL1 protein selected fromthe group consisting of Macro1, Macro 2, and PARP domains; d) anisolated polypeptide consisting essentially of an amino acid sequencethat is at least 70% identical to the amino acid sequence comprisingresidues 453-702 of SEQ ID NO:2 and is not full-length BAL1 and/or whichlacks one or more functional domain(s) of a BAL1 protein selected fromthe group consisting of Macro1, Macro 2, and PARP domains; e) anisolated polypeptide fragment of a BBAP protein which is encoded by anucleic acid molecule comprising a nucleotide sequence encoding at leastone BBAP binding domain and does not encode full-length BAL1; f) anisolated polypeptide fragment of a BAL1 protein which is encoded by anucleic acid molecule comprising a nucleotide sequence encoding at leastone BBAP binding domain and which does not encode one or more functionaldomain(s) selected from the group consisting of Macro1, Macro 2, andPARP domains; g) an isolated polypeptide which is encoded by a nucleicacid molecule comprising a nucleotide sequence encoding an amino acidsequence that is at least 70% identical to the amino acid sequence ofresidues 453-702 of SEQ ID NO:2 and which does not encode full-lengthBAL1 and/or which does not encode one or more functional domain(s) of aBAL1 protein selected from the group consisting of Macro1, Macro 2, andPARP domains; h) an isolated polypeptide which is encoded by a nucleicacid molecule consisting essentially of a nucleotide sequence encodingan amino acid sequence that is at least 70% identical to the amino acidsequence of residues 453-702 of SEQ ID NO:2 and does not encodefull-length BAL1 and/or does not encode one or more functional domain(s)of a BAL1 protein selected from the group consisting of Macro1, Macro 2,and PARP domains; i) an isolated polypeptide fragment of a BBAP proteincomprising at least one BAL1 binding domain and is not full-length BBAP;j) an isolated polypeptide fragment of a BBAP protein comprising atleast one BAL1 binding domain and which lacks one or more functionaldomain(s) selected from the group consisting of BBAP dimerization andRING domains; k) an isolated polypeptide comprising an amino acidsequence that is at least 70% identical to the amino acid sequence ofresidues 423-617 of SEQ ID NO:10 and is not full-length BBAP and/orwhich lacks one or more functional domain(s) of a BBAP protein selectedfrom the group consisting of BBAP dimerization and RING domains; 1) anisolated polypeptide consisting essentially of an amino acid sequencethat is at least 70% identical to the amino acid sequence comprisingresidues 423-617 of SEQ ID NO:10 and is not full-length BBAP and/orwhich lacks one or more functional domain(s) of a BBAP protein selectedfrom the group consisting of BBAP dimerization and RING domains; m) anisolated polypeptide fragment of a BBAP protein which is encoded by anucleic acid molecule comprising a nucleotide sequence encoding at leastone BAL1 binding domain and does not encode full-length BBAP; n) anisolated polypeptide fragment of a BBAP protein which is encoded by anucleic acid molecule comprising a nucleotide sequence encoding at leastone BAL1 binding domain and which does not encode one or more functionaldomain(s) selected from the group consisting of BBAP dimerization andRING domains; o) an isolated polypeptide which is encoded by a nucleicacid molecule comprising a nucleotide sequence encoding an amino acidsequence that is at least 70% identical to the amino acid sequence ofresidues 423-617 of SEQ ID NO:10 and which does not encode full-lengthBBAP and/or which does not encode one or more functional domain(s) of aBBAP protein selected from the group consisting of BBAP dimerization andRING domains; and p) an isolated polypeptide which is encoded by anucleic acid molecule consisting essentially of a nucleotide sequenceencoding an amino acid sequence that is at least 70% identical to theamino acid sequence of residues 423-617 of SEQ ID NO:0 and does notencode full-length BBAP and/or does not encode one or more functionaldomain(s) of a BBAP protein selected from the group consisting of BBAPdimerization and RING domains is provided. In one embodiment, theisolated polypeptide maintains the ability to promote one or morebiological activities selected from the group consisting of a) bindingto a BAL1 polypeptide or fragment thereof; b) binding to a BBAPpolypeptide or fragment thereof; c) forming a BAL1-BBAP complex; d)inhibiting localization and/or binding of BAL1 and/or BBAP to DNA damagesites; e) inhibiting binding of BAL1 to poly(ADP-ribose) (PAR) chains;f) inhibiting BBAP monoubiquitylation of histones; g) inhibitingBBAP-mediated methylation of histones; h) inhibiting localization and/orbinding to DNA damage sites of at least one polypeptide selected fromthe group consisting of 53 BP1, RAP80, BRCA1, ATM, γH2AX, and MDC1; andi) inhibiting DNA damage responses (DDR). In another embodiment, theisolated polypeptide contains one or more conservative amino acidsubstitutions. In still another embodiment, the isolated polypeptidefurther comprises a heterologous polypeptide. In yet another embodiment,a composition comprising the isolated polypeptide is provided and thecomposition can contain, for example, a pharmaceutically acceptableagent selected from the group consisting of excipients, diluents, andcarriers. In another embodiment, the isolated polypeptide is immobilizedon an object selected from the group consisting of a cell, a metal, aresin, a polymer, a ceramic, a glass, a microelectrode, a graphiticparticle, a bead, a gel, a plate, an array, and a capillary tube.

In another aspect, an isolated antibody or antigen binding portionthereof that specifically binds to a polypeptide described herein isprovided. In one embodiment, the isolated antibody or antigen bindingportion thereof is a monoclonal antibody, polyclonal antibody, chimericantibody, humanized antibody, single-chain antibody, antibody fragment,or is detectably labeled.

In still another aspect, a method of making an isolated hybridoma whichproduces an antibody that specifically binds to a polypeptide describedherein is provided, wherein the method comprises a) immunizing a mammalusing a composition comprising said polypeptide or a nucleic acidmolecule encoding said polypeptide; b) isolating splenocytes from theimmunized mammal; c) fusing the isolated splenocytes with animmortalized cell line to form hybridomas; and d) screening individualhybridomas for production of an antibody which specifically binds withsaid polypeptide thereof to isolate the hybridoma. In one embodiment, anantibody produced by such a hybridoma is provided herein.

In yet another aspect, a method for detecting the presence of apolypeptide of claim 62 in a sample comprising a) contacting the samplewith a compound which selectively binds to the polypeptide; and b)determining whether the compound binds to the polypeptide in the sampleto thereby detect the presence of the polypeptide in the sample isprovided. In one embodiment, the compound which binds to the polypeptideis an antibody.

In another aspect, a non-human animal model engineered to express apolypeptide described herein is provided. In one embodiment, thepolypeptide is overexpressed. In another embodiment, the animal is aknock-in or a transgenic animal. In still another embodiment, the animalis a rodent.

In still another aspect, a cell-free assay for screening for compoundswhich bind to a polypeptide described herein or biologically activeportion thereof described herein with a test compound and determiningthe ability of the test compound to bind to the polypeptide orbiologically active portion thereof is provided. In one embodiment, thebinding of the test compound to the polypeptide or biologically activeportion thereof is detected by a method selected from the groupconsisting of a) detection of binding by direct detection of testcompound/polypeptide binding; b) detection of binding using acompetition binding assay; and c) detection of binding using an assayfor measuring activity and/or expression of BAL1, BBAP, and/or aBAL1-BBAP complex.

In yet another aspect, a method for identifying a compound which bindsto a polypeptide described herein or biologically active portion thereofdescribed herein comprising a) contacting the polypeptide orbiologically active portion thereof, or a cell expressing thepolypeptide or biologically active portion thereof, with a testcompound; and b) determining whether the polypeptide or biologicallyactive portion thereof binds to the test compound is provided. In oneembodiment, the binding of the test compound to the polypeptide orbiologically active portion thereof is detected by a method selectedfrom the group consisting of a) detection of binding by direct detectionof test compound/polypeptide binding; b) detection of binding using acompetition binding assay; and c) detection of binding using an assayfor measuring activity and/or expression of BAL1, BBAP, and/or aBAL1-BBAP complex.

In another aspect, an isolated protein complex comprising (a) a BAL1polypeptide described herein and (b) a BBAP polypeptide described hereinis provided. In one embodiment, the BAL1 polypeptide comprises an aminoacid sequence having at least about 70% identity to the amino acidsequence of SEQ ID NO:2. In another embodiment, the BBAP polypeptidecomprises an amino acid sequence having at least about 70% identity tothe amino acid sequence of SEQ ID NO:10. In still another embodiment,the complex is at least about 75% pure by weight as compared to theweight of the total protein in the sample. In yet another embodiment, atleast one polypeptide or fragment thereof of the complex is a fusionprotein and/or is labeled. In another embodiment, the complex isgenerated within a host cell. In still another embodiment, two or morepolypeptides within the complex are covalently linked.

In still another aspect, a composition comprising an isolated proteincomplex described herein is provided.

In yet another aspect, an isolated antibody that has a higher bindingaffinity for an isolated protein complex described herein than for theuncomplexed polypeptides of the complex is provided.

In another aspect, a method for identifying a compound that modulates aBAL1-BBAP protein complex comprising (a) contacting a protein complexcomprising (i) a BAL1 polypeptide and a polypeptide of claim 62 or (ii)a BBAP polypeptide and a polypeptide of claim 62, with a test compound;and (b) assaying the amount or activity of the complex, wherein a changein the amount or activity of the complex in the presence of the testcompound as compared to the amount or activity of the complex in theabsence of the test compound is indicative of a compound that modulatesa BAL1-BBAP protein complex is provided. In one embodiment, activity ofthe BAL1-BBAP protein complex is selected from the group consisting ofa) binding to a BAL1 polypeptide or fragment thereof; b) binding to aBBAP polypeptide or fragment thereof; c) forming a BAL1-BBAP complex; d)inhibiting localization and/or binding of BAL1 and/or BBAP to DNA damagesites; e) inhibiting binding of BAL1 to poly(ADP-ribose) (PAR) chains;f) inhibiting BBAP monoubiquitylation of histones; g) inhibitingBBAP-mediated methylation of histones; h) inhibiting localization and/orbinding to DNA damage sites of at least one polypeptide selected fromthe group consisting of 53 BP1, RAP80, BRCA1, ATM, γH2AX, and MDC1; andi) inhibiting DNA damage responses (DDR).

BRIEF DESCRIPTION OF FIGURES

FIG. 1A through FIG. 1E show that BAL1 and BBAP are recruited to DNAdamage sites. FIG. 1A and FIG. 1B show GFP-BAL1 (FIG. 1A) and GFP-BBAP(FIG. 1B) recruitment to sites of laser microirradiation. Hela cellswere transfected with GFP-BAL1 or BBAP, laser micro-irradiated andsubsequently analyzed at serial timepoints for GFP-fluorescence in theDNA damage site (representative photos, left, kinetics of recruitment,right). Variations in fluorescence intensity (I) were plotted as afunction of time (t). GFP levels at each time point were determined byaveraging values from 10 cells (+standard error) from a representativeexperiment. FIG. 1C shows the kinetics of endogenous BAL1 recruitment tolaser-induced DNA breaks. Endogenous BAL1 is shown at the top; DAPI isshown in the middle; and merged images are shown at the bottom. Imageswere obtained at baseline (0) and at serial timepoints (0.25-60 min)following laser microirradiation. FIG. 1D shows endogenous BBAPlocalization to laser-induced DNA breaks (0, 1 and 4 min). FIG. 1E showsthe kinetics of BAL1 foci formation following γ-irradiation. Hela cellswere treated with low-dose (100 cGy) irradiation and analyzed for BAL1foci at baseline and at serial timepoints (0.5-60 min) thereafter.

FIG. 2 shows that the BAL1 macro domain 2 is required for recruitment toDNA damage sites. BAL1 protein functional domains, including macrodomains 1 and 2), BBAP binding domain (BBD) and the region with partialsequence homology to PARP catalytic domain, are shown in relative order(domain sizes and amino acids positions below). GFP-tagged BAL1constructs are labeled and represented. Mutations in macro domain 1(D126A) and macro domain 2 (IE 326, 327 AA [IE-AA]) are also shown indark black. Representative images of GFP-BAL1 (293T cell) transfectantsat baseline and 2 min following laser microirradiation are shown to theright. The percentage of cells with GFP-BAL1 recruitment tolaser-induced DNA breaks are shown to the far right.

FIG. 3A through FIG. 3F show that BAL1 and BBAP co-localize with PARP1and PAR and physically associate with PAR-n-proteins following DNAdamage. FIG. 3A and FIG. 3B show the co-localization of PARP1, PAR, andBAL1 (FIG. 3A) or PARP1, PAR and BBAP (FIG. 3B) at laser-induced DNAbreaks (2 min following laser microirradiation). FIG. 3C showsco-localization of BAL1 and PAR foci in γ-irradiated cells. Hela cellswere treated with low-dose irradiation (100 cGy) and analyzed for BAL1and PAR foci at baseline and at serial timepoints (0.5-60 min)thereafter. BAL1 is shown at the top; PAR is shown in the middle; andmerged images are shown at the bottom. FIG. 3D showsco-immunoprecipitation of PAR n-proteins and BALE Hela cells wereuntreated or treated with low-dose Dox (50 ng) for 10 min with orwithout PJ-34 pretreatment. Cell lysates were immunoprecipitated (IP)with anti-PARP1, anti-BAL1 or control IgG and immunoblotted withanti-PARP1, -PAR, -BAL1 or -BBAP antibodies. Input whole cell lysateswere similarly analyzed and immunoblotted for actin as a loadingcontrol. Molecular weight markers are shown at the right. FIG. 3E showsthe recruitment of GFP-tagged PARP1, BAL1 and BBAP to laser-inducedbreaks in control cells or cells pretreated with the PARP inhibitor,PJ-34 (2 min following laser microirradiation). FIG. 3F shows thekinetics of GFP-BAL1 recruitment to laser-induced DNA breaks in controlor PJ-34 pre-treated cells. GFP-BAL1 levels at each time point weredetermined by averaging values from 10 cells (+standard error) in arepresentative experiment.

FIG. 4A through FIG. 4G show analyses of co-immunoprecipitated PARP1,BAL1 and BBAP and the interaction between GST-BAL1 and PAR. FIG. 4Ashows co-immunoprecipitation of PAR-n-protein and BAL1 (see FIG. 2D forconstruct details). Hela cells were untreated or treated with low-doseDox (50 ng) for 10 min with or without PJ-34 pretreatment. Cell lysateswere immunoprecipitated (IP) with anti-PARP1, anti-BAL1 or control IgGand immunoblotted with anti-PARP1, -PAR, -BAL1 or -BBAP antibodies.Input whole cell lysates were similarly analyzed and immunoblotted foractin as a loading control. Molecular weight markers are shown at theright. FIG. 4B shows scanning densitometric analysis results of PARP1immunoprecipitates in untreated cells and cells treated with Dox aloneor Dox following PJ-34 pretreatment. PARP1 is shown in the left box.BAL1 co-immunoprecipitated with PARP1 is shown in the middle box. BBAPis shown in the right box. There was only a modest increase in PARP1immunoprecipitated following Dox treatment (1× control, 1.2× Dox-treatedcells). In contrast, there was a 4.8 fold increase in BAL1 and a 1.8×increase in BBAP co-immunoprecipitated with PARP1 in Dox-treated cells.In addition, chemical PARP inhibition (PJ-34 pretreatment) decreased thecoimmunoprecipitation of BAL1 and BBAP with PARP1 in Dox-treated cells.FIG. 4C shows recombinant GST-tagged BAL1 proteins (BAL1 full length andDM [IE-AA and D126A] mutants (see FIG. 1F for construct details).Proteins were synthesized, size-fractionated by NuPAGE and analyzed byCoomassie Blue staining. FIG. 4D shows FLAG-tagged PARP1. FLAG-taggedPARP1 (M2) was purified from whole cell lysate (WL), size-fractionatedby NuPAGE and analyzed by Coomassie Blue staining. FIG. 4E showsimmunoblotting of PAR-n-proteins following PARP1 activation in vitro. Invitro assays included ˜200 ng purified FLAG-tagged PARP1 and 0, 0.5 or 1mM NAD+. Assays were performed in the presence (+) or absence (−) of thePARP inhibitor, PJ-34. Thereafter, samples were size-fractionated andimmunoblotted with anti-PARP1 and anti-PAR antibodies. FLAG-tagged PARP1was ADP-ribosylated in a dose-dependent manner by NAD+. This enzymaticactivity was inhibited by PJ-34, confirming its specificity. FIG. 4Fshows pulldown of PAR-n-FLAG-tagged PARP1 by GST-BAL1. GST-BAL1 (1 μug)was immobilized on glutathione-sepharose 4B beads and incubated withproducts of in vitro PARP1 activation (+ or − NAD+, as in FIG. 4E).After multiple washes, GST-BAL1 bound proteins were size-fractionatedand immunoblotted with anti-PAR and anti-PARP1 antibodies. The productsof the in vitro PARP1 assay (Input, + or − NAD+) were similarlyanalyzed. GST-tagged BAL1 selectively pulled down PAR-modified PARP1 butdid not bind unmodified PARP1 protein. FIG. 4G shows pulldown ofPAR-n-FLAG-tagged PARP1 by GST-BAL1 mutants. Assays including GST-BAL1,or -BAL1 DM were performed as in FIG. 4F. GST-tagged BAL1 pulled downPAR-modified PARP1 in a macro domain-dependent manner.

FIG. 5A through FIG. 5C show recruitment of endogenous PARP1, PAR, BAL1and BBAP to DNA damage sites. FIG. 5A shows recruitment of endogenousPARP1, PAR, BAL1 and BBAP to laser-induced breaks in control or PJ-34pre-treated cells (2 min following laser microirradiation). FIG. 5Bshows depletion of PARP1, BAL1 and BBAP in Hela cells. FIG. 5C showsrecruitment of BAL1, PARP1, BBAP and PAR to laser-induced breaks incontrol cells or cells depleted of PARP1, BAL1 or BBAP (2 min followinglaser microirradiation).

FIG. 6A and FIG. 6B show co-localization of BAL1 and PAR foci inγ-irradiated control Hela cells (FIG. 6A), and Hela cells pretreatedwith PJ-34 (FIG. 6B). Hela cells were treated with low-dose irradiation(100 cGy) and analyzed for BAL1 and PAR foci at baseline and at serialtimepoints (0.5-60 min) thereafter. BAL1 is shown at the top; PAR isshown in the middle; and merged images are shown at the bottom.

FIG. 7A through FIG. 7D show that BAL1 limits the cellular response toDNA damaging agents. FIG. 7A shows that BAL1 depletion augments thecellular response to DNA damaging agents. Hela cells were transfectedwith control or BAL1 siRNAs (siRNA#1, #2), treated with Dox at 50 ng/ml,200 ng/ml, or left untreated for 1-96 hrs and subsequently evaluated byMTS assay. The consequences of BAL1 depletion were most striking incells treated with low-dose Dox (50 ng) (p<0.001, two-way ANOVA). After72 hr of treatment with low-dose Dox (50 ng), cellular proliferation (asassessed by MTS assay) was ˜70-80% lower in BAL1-depleted cells than incontrol RNAi or parental cells. FIG. 7B shows cellular apoptosisfollowing BAL1 depletion and Dox treatment. Parental, control and BAL1siRNA-transfected Hela cells were untreated or treated with Dox at 50ng/ml and 200 ng/ml for 24 hrs and analyzed for apoptosis with AnnexinV/PI staining. Error bars shown in FIG. 7A and FIG. 7B represent thestandard deviation (SD) of the mean for three replicates in arepresentative experiment. FIG. 7C shows the recruitment of GFP-control,GFP-BAL1 or GFP BAL1DM to laser-induced breaks in (5′ UTR-specific) BAL1siRNA knockdown cells, including GFP-control, GFP-BAL1, GFP-BAL1DM,PARP-1, and merged images. FIG. 7D shows apoptosis of BAL1-depleted Helacells depleted with GFP-control, GFP-BAL1 or GFP-BAL1DM and subsequentlytreated with doxorubicin (50 nM) or left untreated. Apoptosis wasassessed with Annexin V/PI staining. Error bars represent the SD of themean for 3 replicates in a representative experiment.

FIG. 8A through FIG. 8F show that PARP-dependent recruitment of BBAP toDNA damage sites is required for early ubiquitin chain formation.Ubiquitylation (conjugated ubiquitin, FK2 immunostaining) and PARP1(FIG. 8A), BBAP (FIG. 8B) and RNF8 (FIG. 8C) recruitment tolaser-induced DNA breaks in control or PJ-34 pre-treated cells (0, 5 and60 min following DNA damage). FK2 immunostaining and PARP1 (FIG. 8D),BBAP (FIG. 8E) and RNF8 (FIG. 8F) in control siRNA or BAL siRNA cells atthe same timepoints following laser microirradiation.

FIG. 9A through FIG. 9C show the kinetics of ubiquitylation and BBAP,RNF8 and PARP1 recruitment to laser-induced breaks. FIG. 9A shows PARP1localization and ubiquitylation (conjugated ubiquitin, FK2immunostaining) at laser-induced breaks in control or PJ-34 pre-treatedHela cells. FIG. 9B and FIG. 9C show BBAP (FIG. 9B) and RNF8 (FIG. 9C)recruitment in cells treated as in FIG. 9A. Assays were performed as inFIG. 7 and analyzed at serial timepoints following lasermicroirradiation (baseline [0] and 5-60 min).

FIG. 10A through FIG. 10C show that PARP1 activation and BAL1/BBAPrecruitment to DNA damage sites are independent of ATM and MDC1. FIG.10A shows recruitment of PARP1, ATM and MDC1 to laser-induced breaks incontrol or PJ-34 pretreated cells (5 min following lasermicroirradiation). FIG. 10B shows depletion of ATM or MDC1 followingsiRNA. Hela cells treated with a scrambled control (SC) or ATM or MDC1siRNA were lysed, size-fractionated and immunoblotted with therespective antibodies and actin (as a loading control). FIG. 10C showsrecruitment of BAL1 and PARP1 to laser-induced breaks in control cellsor cells depleted of ATM or MDC1. Assays were performed and analyzed atserial timepoints following laser microirradiation (baseline [0] and 1-9min).

FIG. 11A through FIG. 11D show functional analyses of PARP1/BAL1/BBAP-and MDC1/RNF8-associated DDRs by comet assay. Comet assays of Hela cellspre-treated with PJ-34 or vehicle alone (FIG. 11A) or control BAL1, MDC1or BAL1 and MDC1 siRNAs (FIG. 11B) or control, BBAP, RNF8 or BBAP andRNF8 siRNAs (FIG. 11D) treated with low-dose irradiation (200 cGy) andanalyzed under alkaline conditions 15 min, 60 min or 24 hr thereafterare shown. FIG. 11C shows depletion of BBAP or RNF8 following siRNA.Hela cells treated with a scrambled control (SC) or BBAP or RNF8 siRNAwere lysed, size-fractionated and immunoblotted with the respectiveantibodies and actin (as a loading control). Comet tail moment (% DNA intail×tail length) determined for 50-100 cells/condition using TriTekComet Score™ software (bar graphs [mean+/−SD] is shown above andrepresentative photographs are shown below). Data are from one of threesimilar experiments.

FIG. 12A through FIG. 12F show that early 53BP1 recruitment to DNAdamage sites requires PARP1, 854 BAL1 and BBAP. FIG. 12A showsrecruitment of PARP1 and 53BP1 to laser-induced breaks in control orPJ-34 treated cells. Images were obtained at baseline (0) and 10-45 30min following laser microirradiation. FIG. 12Bs show PARP1 and 53BP1recruitment to laser-induced breaks in control cells or cells depletedof PARP1, BAL1 or BBAP (via siRNA) (20 min following lasermicroirradiation). FIG. 12C through FIG. 12F show the kinetics of 53BP1and H2AX foci formation following γ-irradiation of control orPJ-34-treated cells (FIG. 12C and FIG. 12D) or control siRNA or BAL1siRNA treated cells (FIG. 12E and FIG. 12F). Cells were treated withPJ-34 or vehicle alone (FIG. 12C and FIG. 12D) or control siRNA or BAL1siRNA (FIG. 12E and FIG. 12F), subjected to low-dose (100 cGy)irradiation and analyzed for 53BP1 and γH2AX foci at baseline and 1-60min thereafter. FIG. 12C and FIG. 12E show the percentage of cellswith >10 foci/nuclei at each time point and condition. Error barsrepresent the standard deviation (SD) of the mean for 3 independentlystained slides for each time point and condition. At the earliesttimepoints following irradiation (0-4 min), the development of repairfoci (percent of cells with >10 foci/nucleus) was compared in controlvs. PJ-34-treated cells and control siRNA vs. BAL siRNA-treated cellswith an ANOVA. FIG. 12D and FIG. 12F show 53BP1 at the top; γH2AX in themiddle; and merged images at the bottom.

FIG. 13A through FIG. 13D show that early RAP80/BRCA1 recruitment to DNAdamage sites requires PARP1, BAL1 and BBAP. PARP1, RAP80 (FIG. 13A) andBRCA1 (FIG. 13B) recruitment to laser-induced breaks in control or PJ-34treated cells (0-60 min following laser microirradiation). RAP80 (FIG.13A) or BRCA1 (FIG. 13B) is shown at the top; PARP1 is shown in themiddle; and merged images are shown at the bottom. FIG. 13C showsrecruitment of PARP1, RAP80, and BRCA1 to laser-induced breaks incontrol cells or cells depleted of PARP1, BAL1, or BBAP (via siRNA) (10min following laser microirradiation). FIG. 13D shows that DNA-damageinduced ubiquitylation and recruitment of 53BP1 and RAP80/BRCA1 occursvia an early PARP1, BAL1 and BBAP-dependent pathway and a laterphosphorylation-dependent ATM/MDC1/RNF8-associated route.

FIG. 14A through FIG. 14D show the kinetics of recruitment ofRAP80/BRCA1 and PARP1 to laser-induced breaks. RAP80 (FIG. 14A and FIG.14B) or BRCA1 (FIG. 14C and FIG. 14D) and PARP1 localization inlaser-induced breaks in control cells (FIG. 14A and FIG. 14C) or cellsdepleted of BAL1 (via siRNA) (FIG. 14B and FIG. 14D). Assays wereperformed as in FIG. 12 and analyzed at serial timepoints followinglaser microirradiation (baseline [0] and 5-60 min).

FIG. 15A and FIG. 15B show in vitro analysis of BBAP ubiquitylationusing lysine-specific ubiquitin. BBAPhis6 was purified from E. coli. andwas incubated with or without E1/E2 ligase and wild-type ubiquitin,K48-only ubiquitin, or K63-only ubiquitin. Thereafter, samples weresize-fractionated and immunoblotted with the following antibodies:anti-ubiquitin, anti-ubiK48, anti-ubiK63 (FIG. 15A) and anti-BBAP (FIG.15B).

FIG. 16 shows a schematic view of several BBAP deletion constructs.

FIG. 17 shows a schematic view of BBAP's BAL1 binding domain based onthe results of co-immunoprecipitation experiments.

FIG. 18 shows a schematic view of BBAP's homodimerization domain basedon the results of co-immunoprecipitation experiments.

FIG. 19 shows a schematic view of the domain structures of BAL1 andBBAP, as well as the BBAP- and BAL1-interacting domains of therespective proteins.

FIG. 20A and FIG. 20B show the results of siRNA-mediated doubleknockdown of BAL1 and BBAP on apoptosis and chemotherapeutic sensitivityof HeLa cells. FIG. 20A shows the protein level of BAL1 and BBAP with orwithout the siRNA-mediated double knockdown. FIG. 20B shows percentageof apoptotic cells after the siRNA-mediated single or double knockdown.

BRIEF DESCRIPTION OF THE TABLES

Table 1 shows a list of representative BAL1 and BBAP nucleic acid andamino acid sequences from various organisms.

Table 2 shows a list of siRNA sequences used to knockdown BAL1, PARP1,ATM, MDC1, and RNF8 expression.

Table 3 shows sequences of oligonucleotides and primers used to generatevarious constructs, including GFP-BAL1, GFP-BBAP, and GFP-PARP1constructs.

Table 4 shows sequences of oligonucleotides and primers used to generaterecombinant BBAP, BAL1, and PARP1 proteins in E. coli.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based in part on the discovery that the macrodomain-containing protein, BAL1, and BBAP, its natural binding partnerand an E3 ligase, link PARP1 activation, ubiquitylation, and DNA repair.It is demonstrated herein that BAL1 and BBAP are overexpressed inchemotherapy-resistant cancer cells (e.g., lymphoma cells) and that BAL1localizes to DNA damage sites by binding to PARP1-generatedpoly(ADP-ribose) (PAR) chains, recruits BBAP to generate a BAL1-BBAPprotein complex, and thus localizes BBAP to DNA damage sites. It isfurther demonstrated that BBAP initiates the recruitment of early-stageDNA damage repair (DDR) factors, such as 53BP1, RAP80, and BRCA1,independent of the ATM/MDC1 DNA repair pathway in part, by selectivelyubiquitylating and modulating methylation of nucleosomes at histone H4.Accordingly, the methods and compositions described herein are capableof enhancing the efficacy of cancer therapies by disrupting DDRmechanisms in cancer cells via modulation of the amount and/or activityof BAL1, BBAP, and/or BAL1-BBAP complexes. In addition, methods andcompositions are described herein to identify agents useful formodulating the amount and/or activity of BAL1, BBAP, and/or BAL1-BBAPcomplexes, methods and compositions are also provided for predictingcancer therapy.

In order that the present invention may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

The term “altered amount” of a marker or “altered level” of a markerrefers to increased or decreased copy number of the marker and/orincreased or decreased expression level of a particular marker gene orgenes in a cancer sample, as compared to the expression level or copynumber of the marker in a control sample. The term “altered amount” of amarker also includes an increased or decreased protein level of a markerin a sample, e.g., a cancer sample, as compared to the protein level ofthe marker in a normal, control sample.

The “amount” of a marker, e.g., expression or copy number of a marker orMCR, or protein level of a marker, in a subject is “significantly”higher or lower than the normal amount of a marker, if the amount of themarker is greater or less, respectively, than the normal level by anamount greater than the standard error of the assay employed to assessamount, and preferably at least twice, and more preferably three, four,five, ten or more times that amount. Alternately, the amount of themarker in the subject can be considered “significantly” higher or lowerthan the normal amount if the amount is at least about two, andpreferably at least about three, four, or five times, higher or lower,respectively, than the normal amount of the marker.

The term “altered level of expression” of a marker refers to anexpression level or copy number of a marker in a test sample e.g., asample derived from a subject suffering from cancer, that is greater orless than the standard error of the assay employed to assess expressionor copy number, and is preferably at least twice, and more preferablythree, four, five or ten or more times the expression level or copynumber of the marker or chromosomal region in a control sample (e.g.,sample from a healthy subject not having the associated disease) andpreferably, the average expression level or copy number of the marker orchromosomal region in several control samples. The altered level ofexpression is greater or less than the standard error of the assayemployed to assess expression or copy number, and is preferably at leasttwice, and more preferably three, four, five or ten or more times theexpression level or copy number of the marker in a control sample (e.g.,sample from a healthy subject not having the associated disease) andpreferably, the average expression level or copy number of the marker inseveral control samples.

The term “altered activity” of a marker refers to an activity of amarker which is increased or decreased in a disease state, e.g., in acancer sample, as compared to the activity of the marker in a normal,control sample. Altered activity of a marker may be the result of, forexample, altered expression of the marker, altered protein level of themarker, altered structure of the marker, or, e.g., an alteredinteraction with other proteins involved in the same or differentpathway as the marker, or altered interaction with transcriptionalactivators or inhibitors.

The term “altered structure” of a marker refers to the presence ofmutations or allelic variants within the marker gene or maker protein,e.g., mutations which affect expression or activity of the marker, ascompared to the normal or wild-type gene or protein. For example,mutations include, but are not limited to substitutions, deletions, oraddition mutations. Mutations may be present in the coding or non-codingregion of the marker.

The term “altered subcellular localization” of a marker refers to themislocalization of the marker within a cell relative to the normallocalization within the cell e.g., within a healthy and/or wild-typecell. An indication of normal localization of the marker can bedetermined through an analysis of subcellular localization motifs knownin the field that are harbored by marker polypeptides.

The term “amino acid” is intended to embrace all molecules, whethernatural or synthetic, which include both an amino functionality and anacid functionality and capable of being included in a polymer ofnaturally-occurring amino acids. Exemplary amino acids includenaturally-occurring amino acids; analogs, derivatives and congenersthereof; amino acid analogs having variant side chains; and allstereoisomers of any of any of the foregoing. The names of the naturalamino acids are abbreviated herein in accordance with therecommendations of IUPAC-IUB.

Unless otherwise specified herein, the terms “antibody” and “antibodies”broadly encompass naturally-occurring forms of antibodies (e.g. IgG,IgA, IgM, IgE) and recombinant antibodies such as single-chainantibodies, chimeric and humanized antibodies and multi-specificantibodies, as well as fragments and derivatives of all of theforegoing, which fragments and derivatives have at least an antigenicbinding site. Antibody derivatives may comprise a protein or chemicalmoiety conjugated to an antibody. The term “antibody” as used hereinalso includes an “antigen-binding portion” of an antibody (or simply“antibody portion”). The term “antigen-binding portion”, as used herein,refers to one or more fragments of an antibody that retain the abilityto specifically bind to an antigen (e.g., BAL1 polypeptide or fragmentthereof or BBAP polypeptide or fragment thereof or BAL1-BBAP complexes).It has been shown that the antigen-binding function of an antibody canbe performed by fragments of a full-length antibody. Examples of bindingfragments encompassed within the term “antigen-binding portion” of anantibody include (i) a Fab fragment, a monovalent fragment consisting ofthe VL, VH, CL and CH1 domains; (ii) a F(ab′)₂ fragment, a bivalentfragment comprising two Fab fragments linked by a disulfide bridge atthe hinge region; (iii) a Fd fragment consisting of the VH and CH1domains; (iv) a Fv fragment consisting of the VL and VH domains of asingle arm of an antibody, (v) a dAb fragment (Ward et al., (1989)Nature 341:544-546), which consists of a VH domain; and (vi) an isolatedcomplementarity determining region (CDR). Furthermore, although the twodomains of the Fv fragment, VL and VH, are coded for by separate genes,they can be joined, using recombinant methods, by a synthetic linkerthat enables them to be made as a single protein chain in which the VLand VH regions pair to form monovalent polypeptides (known as singlechain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; andHuston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; andOsbourn et al. 1998, Nature Biotechnology 16: 778). Such single chainantibodies are also intended to be encompassed within the term“antigen-binding portion” of an antibody. Any VH and VL sequences ofspecific scFv can be linked to human immunoglobulin constant region cDNAor genomic sequences, in order to generate expression vectors encodingcomplete IgG polypeptides or other isotypes. VH and VL can also be usedin the generation of Fab, Fv or other fragments of immunoglobulins usingeither protein chemistry or recombinant DNA technology. Other forms ofsingle chain antibodies, such as diabodies are also encompassed.Diabodies are bivalent, bispecific antibodies in which VH and VL domainsare expressed on a single polypeptide chain, but using a linker that istoo short to allow for pairing between the two domains on the samechain, thereby forcing the domains to pair with complementary domains ofanother chain and creating two antigen binding sites (see e.g.,Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448;Poljak, R. J., et al. (1994) Structure 2:1121-1123). Still further, anantibody or antigen-binding portion thereof may be part of largerimmunoadhesion polypeptides, formed by covalent or noncovalentassociation of the antibody or antibody portion with one or more otherproteins or peptides. Examples of such immunoadhesion polypeptidesinclude use of the streptavidin core region to make a tetrameric scFvpolypeptide (Kipriyanov, S. M., et al. (1995) Human Antibodies andHybridomas 6:93-101) and use of a cysteine residue, a marker peptide anda C-terminal polyhistidine tag to make bivalent and biotinylated scFvpolypeptides (Kipriyanov, S. M., et al. (1994) Mol. Immunol.31:1047-1058). Antibody portions, such as Fab and F(ab′)₂ fragments, canbe prepared from whole antibodies using conventional techniques, such aspapain or pepsin digestion, respectively, of whole antibodies. Moreover,antibodies, antibody portions and immunoadhesion polypeptides can beobtained using standard recombinant DNA techniques, as described herein.Antibodies may be polyclonal or monoclonal; xenogeneic, allogeneic, orsyngeneic; or modified forms thereof (e.g., humanized, chimeric, etc.).Antibodies may also be fully human. The terms “monoclonal antibodies”and “monoclonal antibody composition”, as used herein, refer to apopulation of antibody polypeptides that contain only one species of anantigen binding site capable of immunoreacting with a particular epitopeof an antigen, whereas the term “polyclonal antibodies” and “polyclonalantibody composition” refer to a population of antibody polypeptidesthat contain multiple species of antigen binding sites capable ofinteracting with a particular antigen. A monoclonal antibody compositiontypically displays a single binding affinity for a particular antigenwith which it immunoreacts.

The term “antisense” nucleic acid refers to oligonucleotides whichspecifically hybridize (e.g., bind) under cellular conditions with agene sequence, such as at the cellular mRNA and/or genomic DNA level, soas to inhibit expression of that gene, e.g., by inhibiting transcriptionand/or translation. The binding may be by conventional base paircomplementarity, or, for example, in the case of binding to DNAduplexes, through specific interactions in the major groove of thedouble helix.

The term “BAL” refers to a family of molecules having certain conservedstructural and functional features. The term “family” when referring tothe protein and nucleic acid molecules of the invention is intended tomean two or more proteins or nucleic acid molecules having a commonstructural domain or motif and having sufficient amino acid ornucleotide sequence homology as defined herein. Such family members canbe naturally or non-naturally occurring and can be from either the sameor different species. For example, a family can contain a first proteinof human origin, as well as other, distinct proteins of human origin oralternatively, can contain homologues of non-human origin. Members of afamily may also have common functional characteristics. For example, BALfamily members encode nuclear proteins with multiple N-terminal macrodomains (MACRO) and a C-terminal poly(ADP-ribose) polymerase (PARP)domain (Aguiar et al. (2005) J. Biol. Chem. 280:33756-33765). Macrodomains are so named because of their original identification in thevariant histone, histone macroH2A. In a positioned nucleosome, the macrodomain of histone macroH2A interferes with transcription factor binding,whereas the histone sequences disrupts SWI/SWF nucleosome remodeling(Angelov et al. (2003) Mol. Cell 11:1033-1041). Without being bound bytheory, BAL macro domain-containing proteins are believed to functionlike histone macroH2A by sterically blocking the access of transcriptionfactors and co-activators to specific chromatin regions. BAL1 and twoadditional family members, BAL2 and BAL3, are the only known proteinswith multiple N-terminal macro domains (Aguiar et al. (2000) Blood96:4328-4334 and Aguiar et al. (2005) J. Biol. Chem. 280:33756-33765).BAL family members also include C-terminal regions with similarities tothe PARP catalytic domain and BAL2 and 3, but not BAL1, which catalyzeADP ribosylation (Aguiar et al. (2005) J. Biol. Chem. 280:33756-33765and Hottiger et al. (201 0) Trends Biochem. Sci. 35:208-219).

In addition, the family of BAL proteins comprise at least one “prolinerich domain.” As used herein, the term “proline rich domain” includes anamino acid sequence of about 4-6 amino acid residues in length havingthe general sequence X-Pro-X-X-Pro-X (where X can be any amino acid)(SEQ ID NO: 53). Proline rich domains are usually located in a helicalstructure and bind through hydrophobic interactions to SH3 domains. SH3domains recognize proline rich domains in both forward and reverseorientations. Proline rich domains are described in, for example,Sattler M. et al., Leukemia (1998) 12:637-644, the contents of which areincorporated herein by reference. BAL proteins of the inventionpreferably include at least one proline rich domain, but may contain twoor more Amino acid residues 781-786 of the human BAL and amino acidresidues 748-753 of the murine BAL comprise proline rich domains. Inaddition, BAL protein family members can be identified based on thepresence of at least one “tyrosine phosphorylation site” in the proteinor corresponding nucleic acid molecule. As used herein, the term“tyrosine phosphorylation site” includes an amino acid sequence of about4 amino acid residues in length having the general sequence Tyr-X-X-X(where X can be any amino acid) (SEQ ID NO: 54). The tyrosine in thisdomain is phosphorylated in response to a cellular stimulus, forexample, in response to a hematopoietic growth factor (e.g.,thrombopoietin, erythropoietin, or steel factor) stimulation. Tyrosinephosphorylation of cellular proteins plays a major role in cellsignaling, e.g., hematopoietic cell signaling. Tyrosine phosphorylationsites are described in, for example, Sattler M. et al., Leukemia (1998)12:637-644, the contents of which are incorporated herein by reference.BAL proteins of the invention include at least one or two tyrosinephosphorylation sites, but may contain three or more. Amino acidresidues 392-395 and 495-498 of the human BAL comprise tyrosinephosphorylation sites. It is to be noted that the term can further beused to refer to any combination of features described herein regardingBAL molecules. For example, any combination of sequence composition,percentage identify, sequence length, domain structure, functionalactivity, etc. can be used to describe a BAL molecule of the presentinvention.

The related term “BAL1” refers to a specific BAL family memberoriginally named B-aggressive lymphoma 1 (BAL1) due to itsoverexpression in diffuse large B-cell lymphomas (DLBCL) (Aguiar et al.(2000) Blood 96:4328-4334 and Takeyama et al. (2003) J. Biol. Chem.278:21930-21937). The term is intended to include fragments, variants(e.g., allelic variants) and derivatives thereof. At least six splicevariants encoding three human Ball isoforms exist. The sequence of humanBAL1 transcript variant 1, which encodes the longest of the three humanBall isoforms (i.e., isoform a), is available to the public at theGenBank database under NM_031458.2 and NP_113646.2. The sequence ofhuman BAL1 transcript variant 2 differs in the 5′ untranslated region(UTR) compared to variant 1 while still encoding the same Ball isoform aand the sequences can be found under NM_001146102.1 and NP_001139574.1.The sequence of human BAL1 transcript variant 3 uses an alternativein-frame splice site in the 5′ coding region relative to variant 1resulting in a shorter protein (i.e., isoform b), and can be found underNM_001146103.1 and NP_001139575.1. The sequence of human BAL1 transcriptvariant 4 uses an alternative 5′ UTR and alternative in-frame splicesite in the 5′ coding region relative to variant 1 resulting in isoformb, and can be found under NM_001146104.1 and NP_001139576.1. Thesequence of human BAL1 transcript variant 5 uses an alternative 5′ UTRand alternative in-frame splice site in the 5′ coding region relative tovariant 1 resulting in isoform b, and can be found under NM_001146105.1and NP_001139577.1. The sequence of human BAL1 transcript variant 6 usesan alternative 5′ UTR, 3′ UTR, and 3′coding region relative to variant 1resulting in a frameshift and can be found under NM_001146106.1 andNP_001139578.1. The frameshift results in a human Ball isoform c, whichhas a shorter and distinct C-terminus relative to isoform a. Nucleicacid and polypeptide sequences of BAL1 orthologs in organisms other thanhumans are well known and include, for example, mouse BAL1 (NM_030253.2and NP_084529.1), chimpanzee BAL1 (XM_516693.2 and XP_516693.2), ratBAL1 (XM_221404.4 and XP_221404.4), cow BAL1 (NM_001076828.1 andNP_001070296.1), dog BAL1 (XM_545132.2 and XP_545132.2), and chickenBAL1 (XM_422116.2 and XP_422116.2). In some embodiments, BAL1 does notcatalyze ADP ribosylation. As further indicated in the Examples, BAL1orthologs have high sequence identity and retain common structuraldomains and functions well known in the art (see, Aguiar et al. (2000)Blood 96:4328-4334 and Takeyama et al. (2003) J. Biol. Chem.278:21930-21937). Representative sequences of BAL1 orthologs arepresented below in Table 1. In addition, numerous annotations of BAL1sequences and structural features have previously been described in U.S.Pat. Nos. 6,870,040 and 7,858,742, each of which is incorporated hereinin its entirety by this reference. It is to be noted that the term canfurther be used to refer to any combination of features described hereinregarding BAL1 molecules. For example, any combination of sequencecomposition, percentage identify, sequence length, domain structure,functional activity, etc. can be used to describe a BAL1 molecule of thepresent invention.

The term “BBAP” can refer to a family of molecules having certainconserved structural and functional features. For example, the family ofBBAP proteins comprise at least one “nuclear localization signal.” Asused herein, the term “nuclear localization signal” includes an aminoacid sequence of about 4-20 amino acid residues in length, which servesto direct a protein to the nucleus. Typically, the nuclear localizationsequence is rich in basic amino acids and exemplary sequences are wellknown in the art (Gorlich D. (1998) EMBO 5.17:2721-7) Amino acidresidues 20-26, 462-478, and 475-478 of the human BBAP comprise nuclearlocalization signals. In addition BBAP proteins can be identified basedon the presence of at least one “C3HC4-type zinc finger motif” in theprotein or corresponding nucleic acid molecule. As used herein, the term“C3HC4-type zinc finger motif” includes an amino acid sequence of about40-70 amino acid residues in length and having the general sequenceC-X-(I,V)-C-X(11-30)-C-X-H-X-(F,I,L)-C-X(2)-C-(I,L,M)-X(10-18)-C-P-X-C,where X can be any amino acid (SEQ ID NO: 55). Proteins comprising sucha ring-H2-finger motif are believed to interact with DNA and to beinvolved in diverse functions, including site specific recombination,DNA repair, and transcriptional regulation. The ring-H₂-finger may alsobind zinc/divalent metal ions to form a structure that is involved inspecific protein-protein interactions (similar to the zinc-cysteineclusters of the adenovirus E1 A). Amino acid residues 561-599 of thehuman BBAP comprise a C3HC4-type zinc finger motif.

In some embodiments, however, the term “BBAP” can refer to a specificBBAP family member originally named B-lymphoma and BAL-associatedprotein due to its E3 ligase activity and RING domain-containingC-terminus having near identity to that of Deltex (DTX) family members(Takeyama et al. (2003) J. Biol. Chem. 278:21930-21937). BBAP promotesself-ubiquitylation in a RING finger-dependent manner and canselectively monoubiquitylates histone H4 at lysine 91 (Takeyama et al.(2003) J. Biol. Chem. 278:21930-21937 and Yan et al. (2009) Mol. Cell36:110-120). BBAP also selectively modulated the kinetics of 53BP1accumulation at DNA damage sites based on the disruption ofBBAP-mediated histone H4K91 ubiquitylation and associated loss ofchromatin-associated histone H4K20 methylase and methylated H4K20, since53BP1 localizes to DNA damage sites by binding to methylated H4K20 (Yanet al. (2009) Mol. Cell 36:110-120). The term is intended to includefragments, variants (e.g., allelic variants) and derivatives thereof.The sequence of a representative human BBAP cDNA is available to thepublic at the GenBank database under NM_138287.3 and NP_612144.1.Nucleic acid and polypeptide sequences of BBAP orthologs in organismsother than humans are well known and include, for example, mouse BBAP(NM_001013371.2 and NP_001013389.2), chimpanzee BBAP (XM_526285.2 andXP_526285.1), rat BBAP (XM_573295.2 and XP_573295.2), cow BBAP(XM_592997.4 and XP_592997.2), dog BBAP (XM_535762.2 and XP_535762.2),and chicken BBAP (XM_422114.2 and XP_422114.2). As further indicated inthe Examples, BBAP orthologs have high sequence identity and retaincommon structural domains and functions well known in the art (see,Aguiar et al. (2000) Blood 96:4328-4334; Takeyama et al. (2003) J. Biol.Chem. 278:21930-21937; Monti et al. (2005) Blood 105:1851-1861;Juszczynski et al. (2006) Mol. Cell Biol. 26:5348-5359; and Aguiar etal. (2005) J. Biol. Chem. 280:33756-33765). Representative sequences ofBBAP orthologs are presented below in Table 1. In addition, numerousannotations of BBAP sequences and structural features have previouslybeen described in U.S. Pat. Nos. 7,112,420 and 7,632,660, each of whichis incorporated herein in its entirety by this reference.

The term “binding” or “interacting” refers to an association, which maybe a stable association, between two molecules, e.g., between apolypeptide of the invention and a binding partner, due to, for example,electrostatic, hydrophobic, ionic and/or hydrogen-bond interactionsunder physiological conditions. Exemplary interactions includeprotein-protein, protein-nucleic acid, protein-small molecule, and smallmolecule-nucleic acid interactions.

The term “biological sample” is intended to include tissues, cells andbiological fluids isolated from a subject, as well as tissues, cells andfluids present within a subject.

The term “body fluid” refers to fluids that are excreted or secretedfrom the body as well as fluid that are normally not (e g amnioticfluid, aqueous humor, bile, blood and blood plasma, cerebrospinal fluid,cerumen and earwax, cowper's fluid or pre-ejaculatory fluid, chyle,chyme, stool, female ejaculate, interstitial fluid, intracellular fluid,lymph, menses, breast milk, mucus, pleural fluid, pus, saliva, sebum,semen, serum, sweat, synovial fluid, tears, urine, vaginal lubrication,vitreous humor, vomit). In some embodiments, media described herein cancontain or comprise body fluids.

The terms “cancer” or “tumor” or “hyperproliferative disorder” refer tothe presence of cells possessing characteristics typical ofcancer-causing cells, such as uncontrolled proliferation, immortality,metastatic potential, rapid growth and proliferation rate, and certaincharacteristic morphological features. Cancer cells are often in theform of a tumor, but such cells may exist alone within an animal, or maybe a non-tumorigenic cancer cell, such as a leukemia cell. Cancersinclude, but are not limited to, B cell cancer, e.g., multiple myeloma,Waldenstrom's macroglobulinemia, the heavy chain diseases, such as, forexample, alpha chain disease, gamma chain disease, and mu chain disease,benign monoclonal gammopathy, and immunocytic amyloidosis, melanomas,breast cancer, lung cancer, bronchus cancer, colorectal cancer, prostatecancer, pancreatic cancer, stomach cancer, ovarian cancer, urinarybladder cancer, brain or central nervous system cancer, peripheralnervous system cancer, esophageal cancer, cervical cancer, uterine orendometrial cancer, cancer of the oral cavity or pharynx, liver cancer,kidney cancer, testicular cancer, biliary tract cancer, small bowel orappendix cancer, salivary gland cancer, thyroid gland cancer, adrenalgland cancer, osteosarcoma, chondrosarcoma, cancer of hematologicaltissues, and the like. Other non-limiting examples of types of cancersapplicable to the methods encompassed by the present invention includehuman sarcomas and carcinomas, e.g., fibrosarcoma, myxosarcoma,liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, colorectal cancer, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, liver cancer,choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervicalcancer, bone cancer, brain tumor, testicular cancer, lung carcinoma,small cell lung carcinoma, bladder carcinoma, epithelial carcinoma,glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma,pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma,meningioma, melanoma, neuroblastoma, retinoblastoma; leukemias, e.g.,acute lymphocytic leukemia and acute myelocytic leukemia (myeloblastic,promyelocytic, myelomonocytic, monocytic and erythroleukemia); chronicleukemia (chronic myelocytic (granulocytic) leukemia and chroniclymphocytic leukemia); and polycythemia vera, lymphoma (Hodgkin'sdisease and non-Hodgkin's disease), multiple myeloma, Waldenstrom'smacroglobulinemia, and heavy chain disease. In some embodiments, thecancer whose phenotype is determined by the method of the invention isan epithelial cancer such as, but not limited to, bladder cancer, breastcancer, cervical cancer, colon cancer, gynecologic cancers, renalcancer, laryngeal cancer, lung cancer, oral cancer, head and neckcancer, ovarian cancer, pancreatic cancer, prostate cancer, or skincancer. In other embodiments, the cancer is breast cancer, prostatecancer, lung cancer, or colon cancer. In still other embodiments, theepithelial cancer is non-small-cell lung cancer, nonpapillary renal cellcarcinoma, cervical carcinoma, ovarian carcinoma (e.g., serous ovariancarcinoma), or breast carcinoma. The epithelial cancers may becharacterized in various other ways including, but not limited to,serous, endometrioid, mucinous, clear cell, brenner, orundifferentiated. In some embodiments, the present invention is used inthe treatment, diagnosis, and/or prognosis of lymphoma or its subtypes,including, but not limited to, lymphocyte-rich classical Hodgkinlymphoma, mixed cellularity classical Hodgkin lymphoma,lymphocyte-depleted classical Hodgkin lymphoma, nodular sclerosisclassical Hodgkin lymphoma, anaplastic large cell lymphoma, diffuselarge B-cell lymphomas, MLL⁺ pre B-cell ALL) based upon analysis ofmarkers described herein.

The term “complex” refers to an association between at least twomoieties (e.g. chemical or biochemical) that have an affinity for oneanother. “Protein complex” or “polypeptide complex” refers to a complexcomprising at least one polypeptide. In one embodiment, a complexcomprises a BAL1 polypeptide or fragment thereof along with a BBAPpolypeptide or fragment thereof. In another embodiment, a complexcomprises a BAL1 polypeptide and a BAL1-binding domain of a BBAPpolypeptide. In still another embodiment, a complex comprises a BBAPpolypeptide and a BBAP-binding domain of a BAL1 polypeptide. Embodimentsof complexes described herein can encompass other molecules (e.g.,polypeptides) that can bind to the complex, such as an antibody.

As used herein, the term “diagnostic marker” includes markers describedherein which are useful in the diagnosis of cancer, e.g., over- orunder-activity, emergence, expression, growth, remission, recurrence orresistance of tumors before, during or after therapy. The predictivefunctions of the marker may be confirmed by, e.g., (1) increased ordecreased copy number (e.g., by FISH, FISH plus SKY, single-moleculesequencing, e.g., as described in the art at least at J. Biotechnol.,86:289-301, or qPCR), overexpression or underexpression (e.g., by ISH,Northern Blot, or qPCR), increased or decreased protein level (e.g., byIHC), or increased or decreased activity (determined by, for example,modulation of a pathway in which the marker is involved), e.g., in morethan about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%,or more of human cancers types or cancer samples; (2) its presence orabsence in a biological sample, e.g., a sample containing tissue, wholeblood, serum, plasma, buccal scrape, saliva, cerebrospinal fluid, urine,stool, or bone marrow, from a subject, e.g. a human, afflicted withcancer; (3) its presence or absence in clinical subset of subjects withcancer (e.g., those responding to a particular therapy or thosedeveloping resistance). Diagnostic markers also include “surrogatemarkers,” e.g., markers which are indirect markers of cancerprogression.

The term “DNA damage” refers to chemical and/or physical modification ofthe DNA in a cell, including, but not limited to, methylation,alkylation double-stranded breaks, cross-linking, thymidine dimerscaused by ultraviolet light, and oxidative lesions formed by oxygenradical binding to DNA bases. DNA damage initiates a tightly regulatedsignaling cascade and the orderly recruitment of repair factors todamage sites as described further below. The chromatin substrate for DNAdamage repair (DDR), DNA encircling nucleosomes comprised of corehistone proteins, can be modulated in multiple ways —incorporation ofhistone variants, post-translational modification of select histones,repositioning of nucleosomes and generation of DNA repair foci (Polo andJackson (2011) Genes Dev. 25:409-433). Cells utilize specific factors todetect and repair DNA single-strand breaks (SSB) and two complementarypathways, homologous recombination (HR) and non-homologous end-joining(NHEJ), to address double-strand breaks (DSB) (Polo and Jackson (2011)Genes Dev. 25:409-433). One of the earliest responses to single-strandand double-strand DNA breaks is the activation and recruitment ofpoly(ADP-ribose) polymerase protein (PARP) family members. Although thePARP family includes 16 proteins, thus far, only PARP1 and PARP2 havebeen linked to DNA damage responses (Ciccia and Elledge (2010) Mol. Cell40:179-204). Upon activation, PARP1 catalyzes the NAD⁺-dependentaddition of poly(ADP-ribose) (PAR) chains to target proteins includingcertain histones and PARP1 itself. PARP1 activation and associated PARsynthesis occur within seconds of DNA damage and persist for minutes(Polo and Jackson (2011) Genes Dev. 25:409-433). The rapid andshort-lived PARylation at DNA damage sites is thought to promote a morerelaxed chromatin structure which facilitates DNA repair (Krishnakumarand Kraus (2010) Mol. Cell 39:8-24). DDR proteins assemble in acoordinated, sequential manner at sites of DNA breaks (Polo and Jackson(2011) Genes Dev. 25:409-433). The initial recruitment phase is rapid,transient and dependent upon PARylation at DNA damage sites (Polo andJackson (2011) Genes Dev. 25:409-433). A second phase, which also beginswithin seconds but lasts for hours, includes the sequentialphosphorylation and ubiquitylation of multiple DSB repair factors (Poloand Jackson (2011) Genes Dev. 25:409-433). Following the initialrecruitment of the MRN (Mrell, RAD50 and Nbs1) complex, HR DSB repairinvolves ATM localization and phosphorylation of γH2AX and MDC1 (Poloand Jackson (2011) Genes Dev. 25:409-433). ATM-mediated phosphorylationof MDC1 promotes the recruitment of the RNF8 E3 ligase, which targetsH2A histones (Polo and Jackson (2011) Genes Dev. 25:409-433). A secondE3 ligase, RNF168, interacts with ubiquitylated H2A-type histones in aRNF8-dependent manner and amplifies the local concentration of ubiquitinconjugates (Doil et al. (2009) Cell 136:435-446 and Polo and Jackson(2011) Genes Dev. 25:409-433). Of note, RNF8/RNF168 also regulate theretention of the checkpoint mediators, 53BP1 and BRCA 1, at sites of DNAdamage. The mechanisms of RNF8/RNF168-modulated recruitment of 53BP1remain undefined, whereas BRCA 1 localizes to DNA breaks via RAP80, anadaptor protein with ubiquitin interacting motifs (UIM) (Doil et al.(2009) Cell 136:435-446). In some embodiments, “DNA damage repair” or“DDR” refers to one or more of the DNA damage repair processes describedabove. In other embodiments, “DNA repair” refers to a collection ofmechanisms used to repair damage to DNA. A non-limiting list ofexemplary DNA repair mechanisms includes non-homologous end joining(NHEJ), homologous recombination (HR), single-strand break repair,nucleotide excision repair (NER), base excision repair (BER), mismatchexcision repair (MER), and other repair mechanisms using DNApolymerases, editing and processing nucleases and DNA repair helicases.Many genes and genetic elements in mammals (e.g., humans) are well knownto those of skill in the art and are available in such compiled forms asWood et al., Human DNA Repair Genes, Science, 291: 1284-1289 (February2001) and Bulman et al., Locations of DNA Damage Response and RepairGenes in the Mouse and Correlation with Cancer Risk Modifiers, NationalRadiological Protection Board Report, October 2004 (ISBN 0-85951-544-3).In addition, a mouse DNA repair gene database is publicly available atthe UK Health Protection Agency website. Exemplary proteins mediatingNHEJ include, but are not limited to, Ligase4, XRCC4, H2AX, DNAPKcs(DNA-PK), Ku70, Ku80, Artemis, Cernunnos/XLF, MRE11, NBS1, and RAD50.Exemplary homologous recombination proteins include RAD51, RAD52, RAD54,XRCC3, RAD51C, BRCA1, BRCA2 (FANCD1), FANCA, FANCB, FANCC, FANCD2,FANCE, FANCF, FANCG, FANCJ (BRIP1/BACH1), FANCL, and FANCM. Exemplaryproteins mediating BER include, but are not limited to, ung, smug1,mbd4, tdg, off1, myh, nth1, mpg, ape1, ape2, lig3, xrcc1, adprt, adprt12and adprt13. Exemplary proteins mediating MER include, but are notlimited to, msh2, msh3, msh4, msh5, msh6, pms1, pms3, mlh1, mlh3, pms213and pms214. Exemplary DNA repair helicases include BLM and WRN.

As used herein, the term “inhibit” includes the decrease, limitation, orblockage, of, for example a particular action, function, or interaction.For example, cancer is “inhibited” if at least one symptom of thecancer, such as hyperproliferative growth, is alleviated, terminated,slowed, or prevented. As used herein, cancer is also “inhibited” ifrecurrence or metastasis of the cancer is reduced, slowed, delayed, orprevented.

The term “isolated polypeptide” refers to a polypeptide, in certainembodiments prepared from recombinant DNA or RNA, or of syntheticorigin, or some combination thereof, which (1) is not associated withproteins that it is normally found within nature, (2) is isolated fromthe cell in which it normally occurs, (3) is isolated free of otherproteins from the same cellular source, (4) is expressed by a cell froma different species, or (5) does not occur in nature.

The terms “label” or “labeled” refer to incorporation or attachment,optionally covalently or non-covalently, of a detectable marker into amolecule, such as a polypeptide. Various methods of labelingpolypeptides are known in the art and may be used. Examples of labelsfor polypeptides include, but are not limited to, the following:radioisotopes, fluorescent labels, heavy atoms, enzymatic labels orreporter genes, chemiluminescent groups, biotinyl groups, predeterminedpolypeptide epitopes recognized by a secondary reporter (e.g., leucinezipper pair sequences, binding sites for secondary antibodies, metalbinding domains, epitope tags). Examples and use of such labels aredescribed in more detail below. In some embodiments, labels are attachedby spacer arms of various lengths to reduce potential steric hindrance.

The term “marker” refers to any nucleic acid or protein sequencedescribed herein useful for enhancing the efficacy of cancer therapies.In some embodiments, the markers of the present invention correspond toBAL1, BBAP, and/or BAL1-BBAP complexes described herein. Markersdescribed herein include diagnostic, prognostic, and therapeuticmarkers.

The “normal” level of expression of a marker is the level of expressionof the marker in cells of a subject, e.g., a human subject, notafflicted with a cancer, e.g., lung, ovarian, pancreatic, liver, breast,prostate, and colon carcinomas, as well as melanoma and multiplemyeloma. An “over-expression” or “significantly higher level ofexpression” of a marker refers to an expression level in a test samplethat is greater than the standard error of the assay employed to assessexpression, and is preferably at least twice, and more preferably three,four, five or ten times the expression level of the marker in a controlsample (e.g., sample from a healthy subjects not having the markerassociated disease) and preferably, the average expression level of themarker in several control samples. A “significantly lower level ofexpression” of a marker refers to an expression level in a test samplethat is at least twice, and more preferably three, four, five or tentimes lower than the expression level of the marker in a control sample(e.g., sample from a healthy subject not having the marker associateddisease) and preferably, the average expression level of the marker inseveral control samples.

An “overexpression” or “significantly higher level of expression or copynumber” of a marker refers to an expression level or copy number in atest sample that is greater than the standard error of the assayemployed to assess expression or copy number, and is preferably at leasttwice, and more preferably three, four, five or ten or more times theexpression level or copy number of the marker in a control sample (e.g.,sample from a healthy subject not afflicted with cancer) and preferably,the average expression level or copy number of the marker in severalcontrol samples.

The term “response to cancer therapy” relates to any response of thehyperproliferative disorder (e.g., cancer) to a cancer therapy,preferably to a change in tumor mass and/or volume after initiation ofneoadjuvant or adjuvant chemotherapy. Hyperproliferative disorderresponse may be assessed, for example for efficacy or in a neoadjuvantor adjuvant situation, where the size of a tumor after systemicintervention can be compared to the initial size and dimensions asmeasured by CT, PET, mammogram, ultrasound or palpation. Response mayalso be assessed by caliper measurement or pathological examination ofthe tumor after biopsy or surgical resection. Responses may be recordedin a quantitative fashion like percentage change in tumor volume or in aqualitative fashion like “pathological complete response” (pCR),“clinical complete remission” (cCR), “clinical partial remission” (cPR),“clinical stable disease” (cSD), “clinical progressive disease” (cPD) orother qualitative criteria. Assessment of hyperproliferative disorderresponse may be done early after the onset of neoadjuvant or adjuvanttherapy, e.g., after a few hours, days, weeks or preferably after a fewmonths. A typical endpoint for response assessment is upon terminationof neoadjuvant chemotherapy or upon surgical removal of residual tumorcells and/or the tumor bed. This is typically three months afterinitiation of neoadjuvant therapy. In some embodiments, clinicalefficacy of the therapeutic treatments described herein may bedetermined by measuring the clinical benefit rate (CBR). The clinicalbenefit rate is measured by determining the sum of the percentage ofpatients who are in complete remission (CR), the number of patients whoare in partial remission (PR) and the number of patients having stabledisease (SD) at a time point at least 6 months out from the end oftherapy. The shorthand for this formula is CBR=CR+PR+SD over 6 months.In some embodiments, the CBR for a particular cancer therapeutic regimenis at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, or more. Additional criteria for evaluating the response to cancertherapies are related to “survival,” which includes all of thefollowing: survival until mortality, also known as overall survival(wherein said mortality may be either irrespective of cause or tumorrelated); “recurrence-free survival” (wherein the term recurrence shallinclude both localized and distant recurrence); metastasis freesurvival; disease free survival (wherein the term disease shall includecancer and diseases associated therewith). The length of said survivalmay be calculated by reference to a defined start point (e.g., time ofdiagnosis or start of treatment) and end point (e.g., death, recurrenceor metastasis). In addition, criteria for efficacy of treatment can beexpanded to include response to chemotherapy, probability of survival,probability of metastasis within a given time period, and probability oftumor recurrence. For example, in order to determine appropriatethreshold values, a particular cancer therapeutic regimen can beadministered to a population of subjects and the outcome can becorrelated to BAL1, BBAP, and/or BAL1-BBAP complex measurements thatwere determined prior to administration of any cancer therapy. Theoutcome measurement may be pathologic response to therapy given in theneoadjuvant setting. Alternatively, outcome measures, such as overallsurvival and disease-free survival can be monitored over a period oftime for subjects following cancer therapy for whom BAL1, BBAP, and/orBAL1-BBAP complex measurement values are known. In certain embodiments,the same doses of cancer therapeutic agents are administered to eachsubject. In related embodiments, the doses administered are standarddoses known in the art for cancer therapeutic agents. The period of timefor which subjects are monitored can vary. For example, subjects may bemonitored for at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35,40, 45, 50, 55, or 60 months. BAL1, BBAP, and/or BAL1-BBAP complexmeasurement threshold values that correlate to outcome of a cancertherapy can be determined using methods such as those described in theExamples section.

The term “resistance” refers to an acquired or natural resistance of acancer sample or a mammal to a cancer therapy (i.e., being nonresponsiveto or having reduced or limited response to the therapeutic treatment),such as having a reduced response to a therapeutic treatment by 25% ormore, for example, 30%, 40%, 50%, 60%, 70%, 80%, or more, to 2-fold,3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold or more. The reductionin response can be measured by comparing with the same cancer sample ormammal before the resistance is acquired, or by comparing with adifferent cancer sample or a mammal who is known to have no resistanceto the therapeutic treatment. A typical acquired resistance tochemotherapy is called “multidrug resistance.” The multidrug resistancecan be mediated by P-glycoprotein or can be mediated by othermechanisms, or it can occur when a mammal is infected with amulti-drug-resistant microorganism or a combination of microorganisms.The determination of resistance to a therapeutic treatment is routine inthe art and within the skill of an ordinarily skilled clinician, forexample, can be measured by cell proliferative assays and cell deathassays as described herein as “sensitizing.” In some embodiments, theterm “reverses resistance” means that the use of a second agent incombination with a primary cancer therapy (e.g., chemotherapeutic orradiation therapy) is able to produce a significant decrease in tumorvolume at a level of statistical significance (e.g., p<0.05) whencompared to tumor volume of untreated tumor in the circumstance wherethe primary cancer therapy (e.g., chemotherapeutic or radiation therapy)alone is unable to produce a statistically significant decrease in tumorvolume compared to tumor volume of untreated tumor. This generallyapplies to tumor volume measurements made at a time when the untreatedtumor is growing log rhythmically.

An “RNA interfering agent” as used herein, is defined as any agent whichinterferes with or inhibits expression of a target gene, e.g., a markerof the invention, by RNA interference (RNAi). Such RNA interferingagents include, but are not limited to, nucleic acid molecules includingRNA molecules which are homologous to the target gene, e.g., a marker ofthe invention, or a fragment thereof, short interfering RNA (siRNA), andsmall molecules which interfere with or inhibit expression of a targetgene by RNA interference (RNAi). “RNA interference (RNAi)” is anevolutionally conserved process whereby the expression or introductionof RNA of a sequence that is identical or highly similar to a targetgene results in the sequence specific degradation or specificpost-transcriptional gene silencing (PTGS) of messenger RNA (mRNA)transcribed from that targeted gene (see Coburn, G. and Cullen, B.(2002) J. of Virology 76(18):9225), thereby inhibiting expression of thetarget gene. In one embodiment, the RNA is double stranded RNA (dsRNA).This process has been described in plants, invertebrates, and mammaliancells. In nature, RNAi is initiated by the dsRNA-specific endonucleaseDicer, which promotes processive cleavage of long dsRNA intodouble-stranded fragments termed siRNAs. siRNAs are incorporated into aprotein complex that recognizes and cleaves target mRNAs. RNAi can alsobe initiated by introducing nucleic acid molecules, e.g., syntheticsiRNAs or RNA interfering agents, to inhibit or silence the expressionof target genes. As used herein, “inhibition of target gene expression”or “inhibition of marker gene expression” includes any decrease inexpression or protein activity or level of the target gene (e.g., amarker gene of the invention) or protein encoded by the target gene,e.g., a marker protein of the invention. The decrease may be of at least30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or more as compared to theexpression of a target gene or the activity or level of the proteinencoded by a target gene which has not been targeted by an RNAinterfering agent. “Short interfering RNA” (siRNA), also referred toherein as “small interfering RNA” is defined as an agent which functionsto inhibit expression of a target gene, e.g., by RNAi. An siRNA may bechemically synthesized, may be produced by in vitro transcription, ormay be produced within a host cell. In one embodiment, siRNA is a doublestranded RNA (dsRNA) molecule of about 15 to about 40 nucleotides inlength, preferably about 15 to about 28 nucleotides, more preferablyabout 19 to about 25 nucleotides in length, and more preferably about19, 20, 21, or 22 nucleotides in length, and may contain a 3′ and/or 5′overhang on each strand having a length of about 0, 1, 2, 3, 4, or 5nucleotides. The length of the overhang is independent between the twostrands, i.e., the length of the overhang on one strand is not dependenton the length of the overhang on the second strand. Preferably the siRNAis capable of promoting RNA interference through degradation or specificpost-transcriptional gene silencing (PTGS) of the target messenger RNA(mRNA). In another embodiment, an siRNA is a small hairpin (also calledstem loop) RNA (shRNA). In one embodiment, these shRNAs are composed ofa short (e.g., 19-25 nucleotide) antisense strand, followed by a 5-9nucleotide loop, and the analogous sense strand. Alternatively, thesense strand may precede the nucleotide loop structure and the antisensestrand may follow. These shRNAs may be contained in plasmids,retroviruses, and lentiviruses and expressed from, for example, the polIII U6 promoter, or another promoter (see, e.g., Stewart, et al. (2003)RNA April; 9(4):493-501 incorporated by reference herein). RNAinterfering agents, e.g., siRNA molecules, may be administered to asubject having or at risk for having cancer, to inhibit expression of amarker gene of the invention, e.g., a marker gene which is overexpressedin cancer (such as the markers listed in Table 3) and thereby treat,prevent, or inhibit cancer in the subject.

The term “sensitize” means to alter cancer cells or tumor cells in a waythat allows for more effective treatment of the associated cancer with acancer therapy (e.g., chemotherapeutic or radiation therapy. In someembodiments, normal cells are not affected to an extent that causes thenormal cells to be unduly injured by the cancer therapy (e.g.,chemotherapy or radiation therapy). An increased sensitivity or areduced sensitivity to a therapeutic treatment is measured according toa known method in the art for the particular treatment and methodsdescribed herein below, including, but not limited to, cellproliferative assays (Tanigawa N, Kern D H, Kikasa Y, Morton D L, CancerRes 1982; 42: 2159-2164), cell death assays (Weisenthal L M, Shoemaker RH, Marsden J A, Dill P L, Baker J A, Moran E M, Cancer Res 1984; 94:161-173; Weisenthal L M, Lippman M E, Cancer Treat Rep 1985; 69:615-632; Weisenthal L M, In: Kaspers G J L, Pieters R, Twentyman P R,Weisenthal L M, Veerman A J P, eds. Drug Resistance in Leukemia andLymphoma. Langhorne, P A: Harwood Academic Publishers, 1993: 415-432;Weisenthal L M, Contrib Gynecol Obstet 1994; 19: 82-90). The sensitivityor resistance may also be measured in animal by measuring the tumor sizereduction over a period of time, for example, 6 month for human and 4-6weeks for mouse. A composition or a method sensitizes response to atherapeutic treatment if the increase in treatment sensitivity or thereduction in resistance is 25% or more, for example, 30%, 40%, 50%, 60%,70%, 80%, or more, to 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold,20-fold or more, compared to treatment sensitivity or resistance in theabsence of such composition or method. The determination of sensitivityor resistance to a therapeutic treatment is routine in the art andwithin the skill of an ordinarily skilled clinician. It is to beunderstood that any method described herein for enhancing the efficacyof a cancer therapy can be equally applied to methods for sensitizinghyperproliferative or otherwise cancerous cells (e.g., resistant cells)to the cancer therapy.

The term “synergistic effect” refers to the combined effect of two ormore anticancer agents or chemotherapy drugs can be greater than the sumof the separate effects of the anticancer agents or chemotherapy drugsalone.

As used herein, “subject” refers to any healthy animal, mammal or human,or any animal, mammal or human afflicted with a cancer, e.g., lung,ovarian, pancreatic, liver, breast, prostate, and colon carcinomas, aswell as melanoma and multiple myeloma.

The language “substantially free of chemical precursors or otherchemicals” includes preparations of antibody, polypeptide, peptide orfusion protein in which the protein is separated from chemicalprecursors or other chemicals which are involved in the synthesis of theprotein. In one embodiment, the language “substantially free of chemicalprecursors or other chemicals” includes preparations of antibody,polypeptide, peptide or fusion protein having less than about 30% (bydry weight) of chemical precursors or non-antibody, polypeptide, peptideor fusion protein chemicals, more preferably less than about 20%chemical precursors or non-antibody, polypeptide, peptide or fusionprotein chemicals, still more preferably less than about 10% chemicalprecursors or non-antibody, polypeptide, peptide or fusion proteinchemicals, and most preferably less than about 5% chemical precursors ornon-antibody, polypeptide, peptide or fusion protein chemicals.

As used herein, the term “survival” includes all of the following:survival until mortality, also known as overall survival (wherein saidmortality may be either irrespective of cause or tumor related);“recurrence-free survival” (wherein the term recurrence shall includeboth localized and distant recurrence); metastasis free survival;disease free survival (wherein the term disease shall include cancer anddiseases associated therewith). The length of said survival may becalculated by reference to a defined start point (e.g. time of diagnosisor start of treatment) and end point (e.g. death, recurrence ormetastasis). In addition, criteria for efficacy of treatment can beexpanded to include response to chemotherapy, probability of survival,probability of metastasis within a given time period, and probability oftumor recurrence.

A “tissue-specific” promoter is a nucleotide sequence which, whenoperably linked with a polynucleotide which encodes or specifies a geneproduct, causes the gene product to be produced in a living human cellsubstantially only if the cell is a cell of the tissue typecorresponding to the promoter.

A “transcribed polynucleotide” or “nucleotide transcript” is apolynucleotide (e.g. an mRNA, hnRNA, a cDNA, or an analog of such RNA orcDNA) which is complementary to or homologous with all or a portion of amature mRNA made by transcription of a marker of the invention andnormal post-transcriptional processing (e.g. splicing), if any, of theRNA transcript, and reverse transcription of the RNA transcript.

An “underexpression” or “significantly lower level of expression or copynumber” of a marker refers to an expression level or copy number in atest sample that is greater than the standard error of the assayemployed to assess expression or copy number, but is preferably at leasttwice, and more preferably three, four, five or ten or more times lessthan the expression level or copy number of the marker in a controlsample (e.g., sample from a healthy subject not afflicted with cancer)and preferably, the average expression level or copy number of themarker in several control samples.

There is a known and definite correspondence between the amino acidsequence of a particular protein and the nucleotide sequences that cancode for the protein, as defined by the genetic code (shown below).Likewise, there is a known and definite correspondence between thenucleotide sequence of a particular nucleic acid and the amino acidsequence encoded by that nucleic acid, as defined by the genetic code.

GENETIC CODE Alanine (Ala, A) GCA, GCC, GCG, GCT Arginine (Arg, R) AGA,ACG, CGA, CGC, CGG, CGT Asparagine (Asn, N) AAC, AAT Aspartic acid (Asp,D) GAC, GAT Cysteine (Cys, C) TGC, TGT Glutamic acid (Glu, E) GAA, GAGGlutamine (Gln, Q) CAA, CAG Glycine (Gly, G) GGA, GGC, GGG, GGTHistidine (His, H) CAC, CAT Isoleucine (Ile, I) ATA, ATC, ATT Leucine(Leu, L) CTA, CTC, CTG, CTT, TTA, TTG Lysine (Lys, K) AAA, AAGMethionine (Met, M) ATG Phenylalanine (Phe, F) TTC, TTT Proline (Pro, P)CCA, CCC, CCG, CCT Serine (Ser, S) AGC, AGT, TCA, TCC, TCG, TCTThreonine (Thr, T) ACA, ACC, ACG, ACT Tryptophan (Trp, W) TGG Tyrosine(Tyr, Y) TAC, TAT Valine (Val, V) GTA, GTC, GTG, GTT Termination signal(end) TAA, TAG, TGA

An important and well known feature of the genetic code is itsredundancy, whereby, for most of the amino acids used to make proteins,more than one coding nucleotide triplet may be employed (illustratedabove). Therefore, a number of different nucleotide sequences may codefor a given amino acid sequence. Such nucleotide sequences areconsidered functionally equivalent since they result in the productionof the same amino acid sequence in all organisms (although certainorganisms may translate some sequences more efficiently than they doothers). Moreover, occasionally, a methylated variant of a purine orpyrimidine may be found in a given nucleotide sequence. Suchmethylations do not affect the coding relationship between thetrinucleotide codon and the corresponding amino acid.

In view of the foregoing, the nucleotide sequence of a DNA or RNA codingfor a fusion protein or polypeptide of the invention (or any portionthereof) can be used to derive the fusion protein or polypeptide aminoacid sequence, using the genetic code to translate the DNA or RNA intoan amino acid sequence. Likewise, for fusion protein or polypeptideamino acid sequence, corresponding nucleotide sequences that can encodethe fusion protein or polypeptide can be deduced from the genetic code(which, because of its redundancy, will produce multiple nucleic acidsequences for any given amino acid sequence). Thus, description and/ordisclosure herein of a nucleotide sequence which encodes a fusionprotein or polypeptide should be considered to also include descriptionand/or disclosure of the amino acid sequence encoded by the nucleotidesequence. Similarly, description and/or disclosure of a fusion proteinor polypeptide amino acid sequence herein should be considered to alsoinclude description and/or disclosure of all possible nucleotidesequences that can encode the amino acid sequence.

TABLE 1 SEQ ID NO: 1 Human BAL1 (isoform a) cDNA Sequence    1atggactttt ccatggtggc cggagcagca gcttacaatg aaaaatcagg taggattacc   61tcgctctcac tcttgtttca gaaagtcttt gctcagatct ttcctcagtg gagaaagggg  121aatacagaag aatgtctccc ctacaagtgc tcagagactg gtgctcttgg agaaaactat  181agttggcaaa ttcccattaa ccacaatgac ttcaaaattt taaaaaataa tgagcgtcag  241ctgtgtgaag tcctccagaa taagtttggc tgtatctcta ccctggtctc tccagttcag  301gaaggcaaca gcaaatctct gcaagtgttc agaaaaatgc tgactcctag gatagagtta  361tcagtctgga aagatgacct caccacacat gctgttgatg ctgtggtgaa tgcagccaat  421gaagatcttc tgcatggggg aggcctggcc ctggccctgg taaaagctgg tggatttgaa  481atccaagaag agagcaaaca gtttgttgcc agatatggta aagtgtcagc tggtgagata  541gctgtcacgg gagcagggag gcttccctgc aaacagatca tccatgctgt tgggcctcgg  601tggatggaat gggataaaca gggatgtact ggaaagctgc agagggccat tgtaagtatt  661ctgaattatg tcatctataa aaatactcac attaagacag tagcaattcc agccttgagc  721tctgggattt ttcagttccc tctgaatttg tgtacaaaga ctattgtaga gactatccgg  781gttagtttgc aagggaagcc aatgatgagt aatttgaaag aaattcacct ggtgagcaat  841gaggacccta ctgttgctgc ctttaaagct gcttcagaat tcatcctagg gaagagtgag  901ctgggacaag aaaccacccc ttctttcaat gcaatggtcg tgaacaacct gaccctccag  961attgtccagg gccacattga atggcagacg gcagatgtaa ttgttaattc tgtaaaccca 1021catgatatta cagttggacc tgtggcaaag tcaattctac aacaagcagg agttgaaatg 1081aaatcggaat ttcttgccac aaaggctaaa cagtttcaac ggtcccagtt ggtactggtc 1141acaaaaggat ttaacttgtt ctgtaaatat atataccatg tactgtggca ttcagaattt 1201cctaaacctc agatattaaa acatgcaatg aaggagtgtt tggaaaaatg cattgagcaa 1261aatataactt ccatttcctt tcctgccctt gggactggaa acatggaaat aaagaaggaa 1321acagcagcag agattttgtt tgatgaagtt ttaacatttg ccaaagacca tgtaaaacac 1381cagttaactg taaaatttgt gatctttcca acagatttgg agatatataa ggctttcagt 1441tctgaaatgg caaagaggtc caagatgctg agtttgaaca attacagtgt cccccagtca 1501accagagagg agaaaagaga aaatgggctt gaagctagat ctcctgccat caatctgatg 1561ggattcaacg tggaagagat gtatgaggcc cacgcatgga tccaaagaat cctgagtctc 1621cagaaccacc acatcattga gaataatcat attctgtacc ttgggagaaa ggaacatgac 1681attttgtctc agcttcagaa aacttcaagt gtctccatca cagaaattat cagcccagga 1741aggacagagt tagagattga aggagcccgg gctgacctca ttgaggtggt tatgaacatt 1801gaagatatgc tttgtaaagt acaggaggaa atggcaagga aaaaggagcg aggcctttgg 1861cgctcgttag gacagtggac tattcagcaa caaaaaaccc aagacgaaat gaaagaaaat 1921atcatatttc tgaaatgtcc tgtgcctcca actcaagagc ttctagatca aaagaaacag 1981tttgaaaaat gtggtttgca ggttctaaag gtggagaaga tagacaatga ggtccttatg 2041gctgcctttc aaagaaagaa gaaaatgatg gaagaaaaac tgcacaggca acctgtgagc 2101cataggctgt ttcagcaagt cccataccag ttctgcaatg tggtatgcag agttggcttt 2161caaagaatgt actcgacacc ttgcgatcca aaatacggag ctggcatata cttcaccaag 2221aacctcaaaa acctggcaga gaaggccaag aaaatctctg ctgcagataa gctgatctat 2281gtgtttgagg ctgaagtact cacaggcttc ttctgccagg gacatccgtt aaatattgtt 2341cccccaccac tgagtcctgg agctatagat ggtcatgaca gtgtggttga caatgtctcc 2401agccctgaaa cctttgttat ttttagtggc atgcaggcta tacctcagta tttgtggaca 2461tgcacccagg aatatgtaca gtcacaagat tactcatcag gaccaatgag accctttgca 2521cagcatcctt ggaggggatt cgcaagtggc agccctgttg attaaSEQ ID NO: 2 Human BAL1 (isoform a) Amino Acid Sequence   1mdfsmvagaa ayneksgrit slsllfqkvf aqifpqwrkg nteec1pykc setgalgeny  61swqipinhnd fkilknnerq lcevlqnkfg cistlvspvq egnskslqvf rkmltprie1 121svwkddltth avdavvnaan edllhgggla lalvkaggfe iqeeskqfva rygkvsagei 181avtgagrlpc kqiihavgpr wmewdkqgct gklqraivsi lnyviyknth iktvaipals 241sgifqfplnl ctktivetir vslqgkpmms nlkeihlvsn edptvaafka asefilgkse 301lgqettpsfn amvvnnltlq ivqghiewqt advivnsvnp hditvgpvak silqqagvem 361kseflatkak qfqrsqlvlv tkgfnlfcky iyhvlwhsef pkpqilkham keclekcieq 421nitsisfpal gtgnmeikke taaeilfdev ltfakdhvkh qltvkfvifp tdleiykafs 481semakrskml slnnysvpqs treekrengl earspainlm gfnveemyea hawiqrilsl 541qnhhiiennh ilylgrkehd ilsqlqktss vsiteiispg rteleiegar adlievvmni 601edmlckvqee markkerglw rslgqwtiqq qktqdemken iiflkcpypp tqelldqkkg 661fekcglqvlk vekidnevlm aafqrkkkmm eeklhrqpvs hrlfqqvpyq fcnvvcrvgf 721qrmystpcdp kygagiyftk nlknlaekak kisaadkliy vfeaevltgf fcqghplniv 781ppplspgaid ghdsvvdnvs spetfvifsg mqaipqylwt ctqeyvqsqd yssgpmrpfa 841qhpwrgfasg spvd SEQ ID NO: 3 Human BAL1 (isoform b) cDNA Sequence    1atggactttt ccatggtggc cggagcagca gcttacaatg aaaaatcaga gactggtgct   61cttggagaaa actatagttg gcaaattccc attaaccaca atgacttcaa aattttaaaa  121aataatgagc gtcagctgtg tgaagtcctc cagaataagt ttggctgtat ctctaccctg  181gtctctccag ttcaggaagg caacagcaaa tctctgcaag tgttcagaaa aatgctgact  241cctaggatag agttatcagt ctggaaagat gacctcacca cacatgctgt tgatgctgtg  301gtgaatgcag ccaatgaaga tcttctgcat gggggaggcc tggccctggc cctggtaaaa  361gctggtggat ttgaaatcca agaagagagc aaacagtttg ttgccagata tggtaaagtg  421tcagctggtg agatagctgt cacgggagca gggaggcttc cctgcaaaca gatcatccat  481gctgttgggc ctcggtggat ggaatgggat aaacagggat gtactggaaa gctgcagagg  541gccattgtaa gtattctgaa ttatgtcatc tataaaaata ctcacattaa gacagtagca  601attccagcct tgagctctgg gatttttcag ttccctctga atttgtgtac aaagactatt  661gtagagacta tccgggttag tttgcaaggg aagccaatga tgagtaattt gaaagaaatt  721cacctggtga gcaatgagga ccctactgtt gctgccttta aagctgcttc agaattcatc  781ctagggaaga gtgagctggg acaagaaacc accccttctt tcaatgcaat ggtcgtgaac  841aacctgaccc tccagattgt ccagggccac attgaatggc agacggcaga tgtaattgtt  901aattctgtaa acccacatga tattacagtt ggacctgtgg caaagtcaat tctacaacaa  961gcaggagttg aaatgaaatc ggaatttctt gccacaaagg ctaaacagtt tcaacggtcc 1021cagttggtac tggtcacaaa aggatttaac ttgttctgta aatatatata ccatgtactg 1081tggcattcag aatttcctaa acctcagata ttaaaacatg caatgaagga gtgtttggaa 1141aaatgcattg agcaaaatat aacttccatt tcctttcctg cccttgggac tggaaacatg 1201gaaataaaga aggaaacagc agcagagatt ttgtttgatg aagttttaac atttgccaaa 1261gaccatgtaa aacaccagtt aactgtaaaa tttgtgatct ttccaacaga tttggagata 1321tataaggctt tcagttctga aatggcaaag aggtccaaga tgctgagttt gaacaattac 1381agtgtccccc agtcaaccag agaggagaaa agagaaaatg ggcttgaagc tagatctcct 1441gccatcaatc tgatgggatt caacgtggaa gagatgtatg aggcccacgc atggatccaa 1501agaatcctga gtctccagaa ccaccacatc attgagaata atcatattct gtaccttggg 1561agaaaggaac atgacatttt gtctcagctt cagaaaactt caagtgtctc catcacagaa 1621attatcagcc caggaaggac agagttagag attgaaggag cccgggctga cctcattgag 1681gtggttatga acattgaaga tatgctttgt aaagtacagg aggaaatggc aaggaaaaag 1741gagcgaggcc tttggcgctc gttaggacag tggactattc agcaacaaaa aacccaagac 1801gaaatgaaag aaaatatcat atttctgaaa tgtcctgtgc ctccaactca agagcttcta 1861gatcaaaaga aacagtttga aaaatgtggt ttgcaggttc taaaggtgga gaagatagac 1921aatgaggtcc ttatggctgc ctttcaaaga aagaagaaaa tgatggaaga aaaactgcac 1981aggcaacctg tgagccatag gctgtttcag caagtcccat accagttctg caatgtggta 2041tgcagagttg gctttcaaag aatgtactcg acaccttgcg atccaaaata cggagctggc 2101atatacttca ccaagaacct caaaaacctg gcagagaagg ccaagaaaat ctctgctgca 2161gataagctga tctatgtgtt tgaggctgaa gtactcacag gcttcttctg ccagggacat 2221ccgttaaata ttgttccccc accactgagt cctggagcta tagatggtca tgacagtgtg 2281gttgacaatg tctccagccc tgaaaccttt gttattttta gtggcatgca ggctatacct 2341cagtatttgt ggacatgcac ccaggaatat gtacagtcac aagattactc atcaggacca 2401atgagaccct ttgcacagca tccttggagg ggattcgcaa gtggcagccc tgttgattaaSEQ ID NO: 4 Human BAL1 (isoform b) Amino Acid Sequence   1mdfsmvagaa ayneksetga lgenyswqip inhndfkilk nnerqicevl qnkfgcistl  61vspvqegnsk slqvfrkmit prielsvwkd dltthavdav vnaanedllh ggglalalvk 121aggfeiqees kqfvarygkv sageiavtga grlpckqiih avgprwmewd kqgctgklqr 181aivsilnyvi yknthiktva ipalssgifq fplnlctkti vetirvslqg kpmmsnlkei 241hlvsnedptv aafkaasefi lgkselgqet tpsfnamvvn nltlqivqgh iewqtadviv 301nsvnphditv gpvaksilqq agvemksefl atkakqfqrs qlvlvtkgfn lfckyiyhvl 361whsefpkpqi lkhamkecle kcieqnitsi sfpalgtgnm eikketaaei lfdevltfak 421dhvkhqltvk fvifptdlei ykafssemak rskmlslnny svpqstreek renglearsp 481ainlmgfnve emyeahawiq rilslqnhhi iennhilylg rkehdilsql qktssvsite 541iispgrtele iegaradlie vvmniedmlc kvqeemarkk erglwrslgq wtiqqqktqd 601emkeniiflk cpvpptqell dqkkqfekcg lqvlkvekid nevlmaafqr kkkmmeeklh 661rqpvshrlfq qvpyqfcnvv crvgfqrmys tpcdpkygag iyftknlknl aekakkisaa 721dkliyvfeae vltgffcqgh plnivpppls pgaidghdsv vdnvsspetf vifsgmqaip 781qylwtctqey vqsqdyssgp mrpfaqhpwr gfasgspvdSEQ ID NO: 5 Human BAL1 (isoform c) cDNA Sequence    1atggactttt ccatggtggc cggagcagca gcttacaatg aaaaatcaga gactggtgct   61cttggagaaa actatagttg gcaaattccc attaaccaca atgacttcaa aattttaaaa  121aataatgagc gtcagctgtg tgaagtcctc cagaataagt ttggctgtat ctctaccctg  181gtctctccag ttcaggaagg caacagcaaa tctctgcaag tgttcagaaa aatgctgact  241cctaggatag agttatcagt ctggaaagat gacctcacca cacatgctgt tgatgctgtg  301gtgaatgcag ccaatgaaga tcttctgcat gggggaggcc tggccctggc cctggtaaaa  361gctggtggat ttgaaatcca agaagagagc aaacagtttg ttgccagata tggtaaagtg  421tcagctggtg agatagctgt cacgggagca gggaggcttc cctgcaaaca gatcatccat  481gctgttgggc ctcggtggat ggaatgggat aaacagggat gtactggaaa gctgcagagg  541gccattgtaa gtattctgaa ttatgtcatc tataaaaata ctcacattaa gacagtagca  601attccagcct tgagctctgg gatttttcag ttccctctga atttgtgtac aaagactatt  661gtagagacta tccgggttag tttgcaaggg aagccaatga tgagtaattt gaaagaaatt  721cacctggtga gcaatgagga ccctactgtt gctgccttta aagctgcttc agaattcatc  781ctagggaaga gtgagctggg acaagaaacc accccttctt tcaatgcaat ggtcgtgaac  841aacctgaccc tccagattgt ccagggccac attgaatggc agacggcaga tgtaattgtt  901aattctgtaa acccacatga tattacagtt ggacctgtgg caaagtcaat tctacaacaa  961gcaggagttg aaatgaaatc ggaatttctt gccacaaagg ctaaacagtt tcaacggtcc 1021cagttggtac tggtcacaaa aggatttaac ttgttctgta aatatatata ccatgtactg 1081tggcattcag aatttcctaa acctcagata ttaaaacatg caatgaagga gtgtttggaa 1141aaatgcattg agcaaaatat aacttccatt tcctttcctg cccttgggac tggaaacatg 1201gaaataaaga aggaaacagc agcagagatt ttgtttgatg aagttttaac atttgccaaa 1261gaccatgtaa aacaccagtt aactgtaaaa tttgtgatct ttccaacaga tttggagata 1321tataaggctt tcagttctga aatggcaaag aggtccaaga tgctgagttt gaacaattac 1381agtgtccccc agtcaaccag agaggagaaa agagaaaatg ggcttgaagc tagatctcct 1441gccatcaatc tgatgggatt caacgtggaa gagatgtatg aggcccacgc atggatccaa 1501agaatcctga gtctccagaa ccaccacatc attgagaata atcatattct gtaccttggg 1561agaaaggaac atgacatttt gtctcagctt cagaaaactt caagtgtctc catcacagaa 1621attatcagcc caggaaggac agagttagag attgaaggag cccgggctga cctcattgag 1681gtggttatga acattgaaga tatgctttgt aaagtacagg aggaaatggc aaggaaaaag 1741gagcgaggcc tttggcgctc gttaggacag tggactattc agcaacaaaa aacccaagac 1801gaaatgaaag aaaatatcat atttctgaaa tgtcctgtgc ctccaactca agagcttcta 1861gatcaaaaga aacagtttga aaaatgtggt ttgcaggttc taaaggtgga gaagatagac 1921aatgaggtcc ttatggctgc ctttcaaaga aagaagaaaa tgatggaaga aaaactgcac 1981aggcaacctg tgagccatag gctgtttcag caagtcccat accagttctg caatgtggta 2041tgcagagttg gctttcaaag aatgtactcg acaccttgcg gtaggtgtca atgcctcatc 2101attggggcta ctctgtggaa tttggtgagc tgaSEQ ID NO: 6 Human BAL1 (isoforrn c) Amino Acid Sequence   1mdfsmvagaa ayneksetga lgenyswqip inhndfkilk nnerqlcevl qnkfgcistl  61vspvqegnsk slqvfrkmlt prielsvwkd dltthavdav vnaanedllh ggglalalvk 121aggfeiqees kqfvarygkv sageiavtga grlpckqiih avgprwmewd kqgctgklqr 181aivsilnyvi yknthiktva ipalssgifq fplnlctkti vetirvslqg kpmmsnlkei 241hlvsnedptv aafkaasefi lgkselgqet tpsfnamvvn nltlqivqgh iewqtadviv 301nsvnphditv gpvaksilqq agvemksefl atkakqfqrs qlvlvtkgfn lfckyiyhvl 361whsefpkpqi lkhamkecle kcieqnitsi sfpalgtgnm eikketaaei lfdevltfak 421dhvkhqltvk fvifptdlei ykafssemak rskmlslnny svpqstreek renglearsp 481ainlmgfnve emyeahawiq rilslqnhhi iennhilylg rkehdilsql qktssvsite 541iispgrtele iegaradlie vvmniedmlc kvqeemarkk erglwrslgq wtiqqqktqd 601emkeniiflk cpvpptqell dqkkqfekcg lqvlkvekid nevlmaafqr kkkmmeeklh 661rqpvshrlfq qvpyqfcnvv crvgfqrmys tpcgrcqcli igatlwnlvsSEQ ID NO: 7 Mouse BAL1 cDNA Sequence    1atggcctatt acatggatac atgggcggca gctcccgccg aaagaccagc caacaattct   61cttgaagaac attatagatg gcaaattccc attaaacaca atgtcttcga aattttaaag  121agcaatgaga gtcagctatg tgaagtcctc caaaataagt ttggatgcat ctctaccctg  181agctgtccaa ctctagcagg gagcagctct cctgctcaga gagtcttcag aaggaccctg  241atccctggga tagagttatc tgtctggaag gatgacctta ccagacacgt tgttgatgct  301gtggtgaacg cagccaatga aaaccttttg catggaagtg gcctggccgg aagcttggtg  361aaaactggtg gctttgaaat ccaagaagag agcaaaagaa tcattgccaa cgttggtaaa  421atctcagttg gtggaatcgc tatcaccggt gcggggagac ttccttgcca tttgattatc  481catgcggttg gacctcggtg gacagttacg aacagccaga cagctatcga attactgaaa  541tttgccatta ggaacattct agattatgtc accaaatatg atctacgcat taagacagta  601gcaattccag ccctgagctc tggaattttc cagttccctc tggatttgtg tacaagcata  661attttagaaa ctatccggct ttatttccaa gacaagcaaa tgttcggtaa tttgagagag  721attcatctgg tgagcaatga ggaccccact gttgcgtcct ttaaatccgc ctcagaaagc  781atcctaggga gggacctgag ctcttggggg ggtccagaaa ctgaccctgc ttccaccatg  841actcttcgca tcggccgggg cctgactctc cagattgtcc aaggctgtat tgaaatgcaa  901acaacagatg taattgttaa ttctggatac atgcaggatt ttaaatcagg acgagtggca  961cagtcgattc ttagacaagc aggggttgaa atggaaaagg aacttgacaa ggttaacctg 1021tccacagatt atcaagaggt gtgggtcaca aaaggattta aattgtcctg tcagtatgtc 1081ttccatgtgg catggcattc ccaaatcaac aaataccaga tattgaaaga tgcaatgaag 1141tcctgtctag aaaaatgcct taaaccagat ataaattcca tttcctttcc tgctctcggg 1201acaggattga tggatttgaa gaagagtaca gcagctcaga taatgtttga ggaagttttt 1261gcatttgcta aagagcacaa ggaaaaaacg ctaactgtaa agattgtgat ctttccagta 1321gatgtggaga cgtacaagat tttttatgct gaaatgacaa aaaggtccaa cgagctgaat 1381ctcagcggta atagtggtgc tttagccctg cagtggtcca gtggggagca aagaagaggc 1441ggccttgaag ctggatctcc tgccatcaat ctcatgggtg taaaagtggg agagatgtgt 1501gaggcccagg aatggattga aaggttgctg gtctccctgg accaccacat cattgagaat 1561aatcatattc tctatcttgg gaaaaaagag cacgacgtgc tgtctgagct ccagaccagc 1621acaagagtct ccatttcaga gactgtcagt ccaagaacgg ccactttgga gattaaaggt 1681ccccaggctg acctcattga cgcagttatg aggattgaat gtatgctgtg tgacgttcag 1741gaagaagtgg caggaaaaag ggagaaaaat ctttggagct tgtcaggaca ggggaccaac 1801cagcaagaaa aactggataa aatggaagaa tcgtacacat ttcaacgata cccagcatca 1861ttaactcagg aacttcagga ccgaaagaaa cagtttgaaa agtgtggctt gtgggttgtg 1921caggtggagc agatagacaa taaggtgctg ctggctgcct tccaagagaa gaagaaaatg 1981atggaagaga ggacgccaaa gggatctggg agccaaaggt tgtttcagca ggtcccacat 2041cagttctgca atacggtgtg cagagtcggc ttccacagaa tgtattcgac atcctataac 2101ccagtttatg gagccggcat atatttcacc aagagcctca aaaatctagc agacaaggtc 2161aagaaaacct caagcacaga caagctaatc tatgtgtttg aggcagaagt actcacaggg 2221tccttctgtc agggtaattc ctcaaatatc atccctccac cattgagtcc tggggcctta 2281gatgtcaatg acagcgtagt tgacaatgtt tccagccctg aaaccattgt tgtttttaat 2341ggcatgcagg ccatgcccct gtacttgtgg acttgcacac aggataggac attctcacag 2401catccgatgt ggtcacaggg ctactcatca ggaccaggaa tggtctcttc gctgcagtcc 2461tgggaatggg tcttaaatgg cagctctgtt tagSEQ ID NO: 8 Mouse BAL1 Amino Acid Sequence   1mayymdtwaa apaerpanns leehyrwqip ikhnvfeilk snesqlcevl qnkfgcistl  61scptlagsss paqrvfrrtl ipgielsvwk ddltrhvvda vvnaanenll hgsglagslv 121ktggfeiqee skriianvgk isvggiaitg agrlpchlii havgprwtvt nsqtaiellk 181fairnildyv tkydlriktv aipalssgif qfpldlctsi iletirlyfq dkqmfgnlre 241ihlvsnedpt vasfksases ilgrdlsswg gpetdpastm tlrigrgltl qivqgciemq 301ttdvivnsgy mqdfksgrva qsilrqagve mekeldkvnl stdyqevwvt kgfklscqyv 361fhvawhsqin kyqilkdamk sclekclkpd insisfpalg tglmdlkkst aaqimfeevf 421afakehkekt ltvkivifpv dvetykifya emtkrsneln lsgnsgalal qwssgeqrrg 481gleagspain lmgvkvgemc eaqewierll vsldhhiien nhilylgkke hdvlselqts 541trvsisetvs prtatleikg pqadlidavm riecmlcdvq eevagkrekn lwslsgqgtn 601qqekldkmee sytfqrypas ltqelqdrkk qfekcglwvv qveqidnkvl laafqekkkm 661meertpkgsg sqrlfqqvph qfcntvcrvg fhrmystsyn pvygagiyft kslknladkv 721kktsstdkli yvfeaevltg sfcqgnssni ippplspgal dvndsvvdnv sspetivvfn 781gmqamplylw tctqdrtfsq hpmwsqgyss gpgmvsslqs wewvlngssvSEQ ID NO: 9 Human BBAP cDNA Sequence    1atggcctccc acctgcgccc gccgtccccg ctcctcgtgc gggtgtacaa gtccggcccc   61cgagtacgaa ggaagctgga gagctacttc cagagctcta agtcctcggg cggcggggag  121tgcacggtca gcacccagga acacgaagcc ccgggcacct tccgggtgga gttcagtgaa  181agggcagcta aggagagagt gttgaaaaaa ggagagcacc aaatacttgt tgacgaaaaa  241cctgtgccca ttttcctggt acccactgaa aattcaataa agaagaacac gagacctcaa  301atttcttcac tgacacaatc acaagcagaa acaccgtctg gtgatatgca tcaacatgaa  361ggacatattc ctaatgctgt ggattcctgt ctccaaaaga tctttcttac tgtaacagct  421gacctgaact gtaacctgtt ctccaaagag cagagggcat acataaccac actgtgccct  481agtatcagaa aaatggaagg tcacgatgga attgagaagg tgtgtggtga cttccaagac  541attgaaagaa tacatcaatt tttgagtgag cagttcctgg aaagtgagca gaaacaacaa  601ttttcccctt caatgacaga gaggaagcca ctcagtcagc aggagaggga cagctgcatt  661tctccttctg aaccagaaac caaggcagaa caaaaaagca actattttga agttcccttg  721ccttactttg aatactttaa atatatctgt cctgataaaa tcaactcaat agagaaaaga  781tttggtgtaa acattgaaat ccaggagagt tctccaaata tggtctgttt agatttcacc  841tcaagtcgat caggtgacct ggaagcagct cgtgagtctt ttgctagtga atttcagaag  901aacacagaac ctctgaagca agaatgtgtc tctttagcag acagtaagca ggcaaataaa  961ttcaaacagg aattgaatca ccagtttaca aagctcctta taaaggagaa aggaggcgaa 1021ttaactctcc ttgggaccca agatgacatt tcagctgcca aacaaaaaat ctctgaagct 1081tttgtcaaga tacctgtgaa actatttgct gccaattaca tgatgaatgt aattgaggtt 1141gatagtgccc actataaact tttagaaact gaattactac aggagatatc agagatcgaa 1201aaaaggtatg acatttgcag caaggtttct gagaaaggtc agaaaacctg cattctgttt 1261gaatccaagg acaggcaggt agatctatct gtgcatgctt atgcaagttt catcgatgcc 1321tttcaacatg cctcatgtca gttgatgaga gaagttcttt tactgaagtc tttgggcaag 1381gagagaaagc acttacatca gaccaagttt gctgatgact ttagaaaaag acatccaaat 1441gtacactttg tgctaaatca agagtcaatg actttgactg gtttgccaaa tcaccttgca 1501aaggcgaagc agtatgttct aaaaggagga ggaatgtctt cattggctgg aaagaaattg 1561aaagagggtc atgaaacacc gatggacatt gatagcgatg attccaaagc agcttctccg 1621ccactcaagg gctctgtgag ttctgaggcc tcagaactgg acaagaagga aaagggcatc 1681tgtgtcatct gtatggacac cattagtaac aaaaaagtgc taccaaagtg caagcatgaa 1741ttctgcgccc cttgtatcaa caaagccatg tcatataagc caatctgtcc cacatgccag 1801acttcctatg gtattcagaa aggaaatcag ccagagggaa gcatggtttt cactgtttca 1861agagactcac ttccaggtta tgagtccttt ggcaccattg tgattactta ttctatgaaa 1921gcaggcatac aaacagaaga acacccaaac ccaggaaaga gataccctgg aatacagcga 1981actgcatact tgcctgataa taaggaagga aggaaggttt tgaaactgct ttatagggcc 2041tttgaccaaa agctgatttt tacagtgggg tactctcgcg tattaggagt ctcagatgtc 2101atcacttgga atgatattca ccacaaaaca tcccggtttg gaggaccaga aatgtatggc 2161tatcctgatc cttcttacct gaaacgtgtc aaagaggagc tgaaagccaa aggaattgag 2221taa SEQ ID NO: 10 Human BBAP Amino Acid Sequence   1mashlrppsp llvrvyksgp rvrrklesyf qsskssggge ctvstqehea pgtfrvefse  61raakervlkk gehqilvdek pvpiflvpte nsikkntrpq issltqsqae tpsgdmhqhe 121ghipnavdsc lqkifltvta dlncnlfske qrayittlcp sirkmeghdg iekvcgdfqd 181ierihqflse qfleseqkqq fspsmterkp lsqqerdsci spsepetkae qksnyfevpl 241pyfeyfkyic pdkinsiekr fgvnieiqes spnmvcldft ssrsgdleaa resfasefqk 301nteplkqecv sladskqank fkqelnhqft kllikekgge ltllgtqddi saakqkisea 361fvkipvklfa anymmnviev dsahykllet ellqeiseie krydicskvs ekgqktcilf 421eskdrqvdls vhayasfida fqhascqlmr evlllkslgk erkhlhqtkf addfrkrhpn 481vhfvlnqesm tltglpnhla kakqyvlkgg gmsslagkkl keghetpmdi dsddskaasp 541plkgsvssea seldkkekgi cvicmdtisn kkvlpkckhe fcapcinkam sykpicptcq 601tsygiqkgnq pegsmvftvs rdslpgyesf gtivitysmk agiqteehpn pgkrypgiqr 661taylpdnkeg rkvlkllyra fdqkliftvg ysrvlgvsdv itwndihhkt srfggpemyg 721ypdpsylkrv keelkakgie SEQ ID NO: 11 Mouse BBAP cDNA Sequence    1atggcttcca gtcccgaccc gccgtccccg ctactcgtac ggctgcggga gtccatcccc   61aaggcgcaca ggaagctcga gatatacttc cagagccggg cctcgggagg tggggagtgc  121tctgtccagc ccgtgggccc cagcgccccg gacacctacg aggtgaagtt cctaaaaaaa  181gcagataagg agaaagtgtt gaaaaagagc gaacacgaga tgttggtcca taacaaacct  241gtgaccattg tcctggaaac tactaaaaag ccagtagagg acctgagacc cagactccca  301tccttgacac agccagtgga gacaccaagc tccagacctc cgtccttgac ggggtctctg  361gatgaagcac tttgtgatga catacatccc caggacgggc tcgtttctaa ctctgttgac  421tcagttgtcc aaaagatctt tcttgctgtg accgctgagc tgaactgtga cctgctctct  481aaagagcaga gagcatctat aaccactgtc tgccctcaca tcatcaaaag catggagggt  541agtgatggaa ttaagaaggt gtgtggcaac ttcaaagata ttgaaaagat acatcacttc  601ttgagtgagc agcttttgga acgggagcag aaacggaagg gaagcgagca gaaacggaag  661tgcgccccac agaaacacac acctcccgat gtggagcggg agccccctga tcagagcagt  721attcaagttc ctgtgcttct ccttgaatat ttcaagcatg ttaatccggg tagactagag  781ttcatagagt acaaatttgg tgtaaacatt gaaatccaag ctagttctcc caatatggtc  841actgtaggct tcacctccag cccatttggc aacgtagaag aagcaagtca gtcctttgtc  901agagactttc agaaatgctc gcagtctctg aagcaagatt gtatctcttt agaggagcac  961cagagagcaa aggaggtcag acaggagctg agtcgctgct tcccaaagct cttgataaag 1021ggacagggaa gaacgctgac tctcctcggc tctcctgctg acatttcagc cgccacagaa 1081aaagtctccc aaggtcttgg cctgagacct gtgaaaataa ccgcatctgg gtacacgacg 1141ggcatcgagg tcgattcaac acgctttaag cttctagagc ctgaactgct ccaggaaatc 1201tcagagatcg agcagaagtt taacacccgt ggcaaagtcc aggaaaaagg ccagaaaacc 1261tgcattcttt ttgtccccaa ggataaagac ttagacctgt cagtgcagtc ctacacaggt 1321tttactgatg ccttccagcg tgccacgtgg cagctgagga cagaagttct gtcgctgaaa 1381gggttgggca aggagagagc tcgcttacac aataccaagt ttgccgacaa ctttaaaaaa 1441gagcacccga atgtgcactt tgtgacatct caggagtcag tgaccttgac tggcttgcca 1501catcaccttg cgcaggcaat gcagtatgtc tccaaaagaa tgggactggc accgtcatct 1561ggagagaaac tcgctatgga tcaggaaacc cccatggaga tcagcagtag tgacccccat 1621ggagatcagc aggagaatgc agccttacct gctccccgag gcacctctag cagccctgca 1681gcttcgaagg ggactgagga ctactgtgtc atctgcatgg ataccatcag caacaagcac 1741gtgctcccca agtgcaagca tgaattctgc acctcgtgta tcagcaaagc catgcttatc 1801aagcctgtct gtcctgtgtg tctgacttcc tacggcatcc agaaagggaa ccagccagag 1861ggaaccatgt cttactccac tcaaaaaggg tcacttccag gttatgaagg ctgtggcacc 1921attgtgatta attatgaaat aaaagatggc atccaaacaa aagagcaccc aaacccagga 1981aaggcttatc atggaacacg gcgaactgca tacttgcctg ataatactga gggaagaaag 2041gttttggatc tgctccacga agcctttaag cacagactga ctttcacaat aggatactct 2101cgagcaacag gagtctcgga tgtcattaca tggaatgata ttcatcacaa aacatccaag 2161tttggaggac cagcaaattt tggctaccct gatcctgatt acctgaaacg tgtcaaggag 2221gagctgaaag caaaaggcat tgagtaaSEQ ID NO: 12 Mouse BBAP Amino Acid Sequence   1masspdppsp llvrlresip kahrkleiyf qsrasgggec svqpvgpsap dtyevkflkk  61adkekvlkks ehemlvhnkp vtivlettkk pvedlrprlp sltqpvetps srppsitgsl 121dealcddihp qdglvsnsvd svvqkiflav taelncdlls keqrasittv cphiiksmeg 181sdgikkvcgn fkdiekihhf lseqllereq krkgseqkrk capqkhtppd vereppdqss 241iqvpvllley fkhvnpgrle fieykfgvni eiqasspnmv tvgftsspfg nveeasqsfv 301rdfqkcsqsl kqdcisleeh qrakevrqel srcfpkllik gqgrtltllg spadisaate 361kvsqglglrp vkitasgytt gievdstrfk llepellgei seieqkfntr gkvqekgqkt 421cilfvpkdkd ldlsvqsytg ftdafqratw qlrtevlslk glgkerarlh ntkfadnfkk 481ehpnvhfvts qesvtltglp hhlaqamqyv skrmglapss geklamdget pmeisssdph 541gdqqenaalp aprgtssspa askgtedycv icmdtisnkh vlpkckhefc tsciskamli 601kpvcpvclts ygiqkgnqpe gtmsystqkg slpgyegcgt ivinyeikdg iqtkehpnpg 661kayhgtrrta ylpdntegrk vldllheafk hrltftigys ratgvsdvit wndihhktsk 721fggpanfgyp dpdylkrvke elkakgie

Before the present invention is further described, it will beappreciated that specific sequence identifiers (SEQ ID NOs) have beenreferenced throughout the specification for purposes of illustration andshould therefore not be construed to be limiting. Any marker of theinvention, including, but not limited to, the markers described in thespecification and markers described herein are well known in the art andcan be used in the embodiments of the invention.

It is further to be understood that this invention is not limited toparticular embodiments described, as such may, of course, vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “and,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “aBAL1-BBAP complex” includes a plurality of such complexes and referenceto “the active agent” includes reference to one or more active agentsand equivalents thereof known to those skilled in the art, and so forth.It is further noted that the claims may be drafted to exclude anyoptional element. As such, this statement is intended to serve asantecedent basis for use of such exclusive terminology as “solely,”“only” and the like in connection with the recitation of claim elements,or use of a “negative” limitation.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

I. Isolated Nucleic Acids

One aspect of the invention pertains to methods utilizing isolatednucleic acid molecules that encode BAL1 and/or BBAP or biologicallyactive portions thereof. As used herein, the term “nucleic acidmolecule” is intended to include DNA molecules (i.e., cDNA or genomicDNA) and RNA molecules (i.e., mRNA) and analogs of the DNA or RNAgenerated using nucleotide analogs. The nucleic acid molecule can besingle-stranded or double-stranded, but preferably is double-strandedDNA. An “isolated” nucleic acid molecule is one which is separated fromother nucleic acid molecules which are present in the natural source ofthe nucleic acid. Preferably, an “isolated” nucleic acid is free ofsequences which naturally flank the nucleic acid (i.e., sequenceslocated at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA ofthe organism from which the nucleic acid is derived. For example, invarious embodiments, the isolated BAL1 and/or BBAP nucleic acid moleculecan contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1kb of nucleotide sequences which naturally flank the nucleic acidmolecule in genomic DNA of the cell from which the nucleic acid isderived (i.e., alymphoma cell). Moreover, an “isolated” nucleic acidmolecule, such as a cDNA molecule, can be substantially free of othercellular material, or culture medium when produced by recombinanttechniques, or chemical precursors or other chemicals when chemicallysynthesized.

A nucleic acid molecule of the present invention, e.g., a nucleic acidmolecule having the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, and11 or a nucleotide sequence which is at least about 50%, preferably atleast about 60%, more preferably at least about 70%, yet more preferablyat least about 80%, still more preferably at least about 90%, and mostpreferably at least about 95% or more (e.g., about 98%) homologous tothe nucleotide sequence shown in SEQ ID NOs: 1, 3, 5, 7, 9, and 11 or aportion thereof (i.e., 100, 200, 300, 400, 450, 500, or morenucleotides), can be isolated using standard molecular biologytechniques and the sequence information provided herein. For example, ahuman BAL1 and/or BBAP cDNA can be isolated from a human muscle cellline (from Stratagene, LaJolla, Calif., or Clontech, Palo Alto, Calif.)using all or portion of SEQ ID NOs: 1, 3, 5, 7, 9, or 11, or fragmentthereof, as a hybridization probe and standard hybridization techniques(i.e., as described in Sambrook, J., Fritsh, E. F., and Maniatis, T.Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989). Moreover, a nucleic acid molecule encompassing all or aportion of SEQ ID NOs: 1, 3, 5, 7, 9 or 11 or a nucleotide sequencewhich is at least about 50%, preferably at least about 60%, morepreferably at least about 70%, yet more preferably at least about 80%,still more preferably at least about 90%, and most preferably at leastabout 95% or more homologous to the nucleotide sequence shown in SEQ IDNOs: 1, 3, 5, 7, 9, or 11, or fragment thereof, can be isolated by thepolymerase chain reaction using oligonucleotide primers designed basedupon the sequence of SEQ ID NOs: 1, 3, 5, 7, 9, or 11, or fragmentthereof, or the homologous nucleotide sequence. For example, mRNA can beisolated from muscle cells (i.e., by the guanidinium-thiocyanateextraction procedure of Chirgwin et al. (1979) Biochemistry 18:5294-5299) and cDNA can be prepared using reverse transcriptase (i.e.,Moloney MLV reverse transcriptase, available from Gibco/BRL, Bethesda,Md.; or AMV reverse transcriptase, available from Seikagaku America,Inc., St. Petersburg, Fla.). Synthetic oligonucleotide primers for PCRamplification can be designed based upon the nucleotide sequence shownin SEQ ID NOs: 1, 3, 5, 7, 9, or 11, or fragment thereof, or to thehomologous nucleotide sequence. A nucleic acid of the invention can beamplified using cDNA or, alternatively, genomic DNA, as a template andappropriate oligonucleotide primers according to standard PCRamplification techniques. The nucleic acid so amplified can be clonedinto an appropriate vector and characterized by DNA sequence analysis.Furthermore, oligonucleotides corresponding to a BAL1 and/or BBAPnucleotide sequence can be prepared by standard synthetic techniques,i.e., using an automated DNA synthesizer.

Probes based on the BAL1 and/or BBAP nucleotide sequences can be used todetect transcripts or genomic sequences encoding the same or homologousproteins. In preferred embodiments, the probe further comprises a labelgroup attached thereto, i.e., the label group can be a radioisotope, afluorescent compound, an enzyme, or an enzyme co-factor. Such probes canbe used as a part of a diagnostic test kit for identifying cells ortissue which express a BAL1 and/or BBAP protein, such as by measuring alevel of a BAL1 and/or BBAP-encoding nucleic acid in a sample of cellsfrom a subject, i.e., detecting BAL1 and/or BBAP mRNA levels.

Nucleic acid molecules encoding other BAL1 and/or BBAP members and thuswhich have a nucleotide sequence which differs from the BAL1 and/or BBAPsequences of SEQ ID NOs: 1, 3, 5, 7, 9, or 11, or fragment thereof, arecontemplated. Moreover, nucleic acid molecules encoding BAL1 and/or BBAPproteins from different species, and thus which have a nucleotidesequence which differs from the BAL1 and/or BBAP sequences of SEQ IDNOs: 1, 3 5, 7, 9, or 11 are also intended to be within the scope of thepresent invention. For example, rat or monkey BAL1 and/or BBAP cDNA canbe identified based on the nucleotide sequence of a human and/or mouseBAL1 and/or BBAP. In one embodiment, the nucleic acid molecule(s) of theinvention encodes a protein or portion thereof which includes an aminoacid sequence which is sufficiently homologous to an amino acid sequenceof SEQ ID NO: 2, 4, 6, 8, 10, or 12, or fragment thereof, such that theprotein or portion thereof modulates (e.g., enhance), one or more of thefollowing biological activities: a) binding to a BAL1 polypeptide orfragment thereof; b) binding to a BBAP polypeptide or fragment thereof;c) forming a BAL1-BBAP complex; d) inhibiting localization and/orbinding of BAL1 and/or BBAP to DNA damage sites; e) inhibiting bindingof BAL1 to poly(ADP-ribose) (PAR) chains; f) inhibiting BBAPmonoubiquitylation of histones; g) inhibiting BBAP-mediated methylationof histones; h) inhibiting localization and/or binding to DNA damagesites of at least one polypeptide selected from the group consisting of53 BP1, RAP80, BRCA1, ATM, γH2AX, and MDC1; and i) inhibiting DNA damageresponses (DDR).

As used herein, the language “sufficiently homologous” refers toproteins or portions thereof which have amino acid sequences whichinclude a minimum number of identical or equivalent (e.g., an amino acidresidue which has a similar side chain as an amino acid residue in SEQID NO: 2, 4, 6, 8, 10, or 12, or fragment thereof) amino acid residuesto an amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, or 12, orfragment thereof, such that the protein or portion thereof modulates(e.g., enhance) one or more of the following biological activities: a)binding to a BAL1 polypeptide or fragment thereof; b) binding to a BBAPpolypeptide or fragment thereof; c) forming a BAL1-BBAP complex; d)inhibiting localization and/or binding of BAL1 and/or BBAP to DNA damagesites; e) inhibiting binding of BAL1 to poly(ADP-ribose) (PAR) chains;f) inhibiting BBAP monoubiquitylation of histones; g) inhibitingBBAP-mediated methylation of histones; h) inhibiting localization and/orbinding to DNA damage sites of at least one polypeptide selected fromthe group consisting of 53 BP1, RAP80, BRCA1, ATM, γH2AX, and MDC1; andi) inhibiting DNA damage responses (DDR).

In another embodiment, the protein is at least about 50%, preferably atleast about 60%, more preferably at least about 70%, 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous to theentire amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, or 12, or afragment thereof.

Portions of proteins encoded by the BAL1 and/or BBAP nucleic acidmolecule of the invention are preferably biologically active portions ofthe BAL1 and/or BBAP protein. As used herein, the term “biologicallyactive portion of BAL1 and/or BBAP” is intended to include a portion,e.g., a domain/motif, of BAL1 and/or BBAP that has one or more of thebiological activities of the full-length BAL1 and/or BBAP protein.

Standard binding assays, e.g., immunoprecipitations and yeast two-hybridassays, as described herein, or functional assays, e.g., RNAi oroverexpression experiments, can be performed to determine the ability ofa BAL1 and/or BBAP protein or a biologically active fragment thereof tomaintain a biological activity of the full-length BAL1 and/or BBAPprotein.

The invention further encompasses nucleic acid molecules that differfrom the nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, or 11,or fragment thereof due to degeneracy of the genetic code and thusencode the same BAL1 and/or BBAP protein as that encoded by thenucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, or 11, orfragment thereof. In another embodiment, an isolated nucleic acidmolecule of the invention has a nucleotide sequence encoding a proteinhaving an amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, or 12,or fragment thereof, or fragment thereof, or a protein having an aminoacid sequence which is at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous to the amino acidsequence of SEQ ID NO: 2, 4, 6, 8, 10, or 12, or fragment thereof, or afragment thereof. In another embodiment, a nucleic acid encoding a BAL1and/or BBAP polypeptide consists of nucleic acid sequence encoding aportion of a full-length BAL1 and/or BBAP fragment of interest that isless than 195, 190, 185, 180, 175, 170, 165, 160, 155, 150, 145, 140,135, 130, 125, 120, 115, 110, 105, 100, 95, 90, 85, 80, 75, or 70 aminoacids in length.

It will be appreciated by those skilled in the art that DNA sequencepolymorphisms that lead to changes in the amino acid sequences of BAL1and/or BBAP may exist within a population (e.g., a mammalian and/orhuman population). Such genetic polymorphism in the BAL1 and/or BBAPgene may exist among individuals within a population due to naturalallelic variation. As used herein, the terms “gene” and “recombinantgene” refer to nucleic acid molecules comprising an open reading frameencoding a BAL1 and/or BBAP protein, preferably a mammalian, e.g.,human, BAL1 and/or BBAP protein. Such natural allelic variations cantypically result in 1-5% variance in the nucleotide sequence of the BAL1and/or BBAP gene. Any and all such nucleotide variations and resultingamino acid polymorphisms in BAL1 and/or BBAP that are the result ofnatural allelic variation and that do not alter the functional activityof BAL1 and/or BBAP are intended to be within the scope of theinvention. Moreover, nucleic acid molecules encoding BAL1 and/or BBAPproteins from other species, and thus which have a nucleotide sequencewhich differs from the human or mouse sequences of SEQ ID NO: 1, 3, 5,or 7, are intended to be within the scope of the invention. Nucleic acidmolecules corresponding to natural allelic variants and homologues ofthe human or mouse BAL1 and/or BBAP cDNAs of the invention can beisolated based on their homology to the human or mouse BAL1 and/or BBAPnucleic acid sequences disclosed herein using the human or mouse cDNA,or a portion thereof, as a hybridization probe according to standardhybridization techniques under stringent hybridization conditions (asdescribed herein).

In addition to naturally-occurring allelic variants of the BAL1 and/orBBAP sequence that may exist in the population, the skilled artisan willfurther appreciate that changes can be introduced by mutation into thenucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, or 11, or fragmentthereof, thereby leading to changes in the amino acid sequence of theencoded BAL1 and/or BBAP protein, without altering the functionalability of the BAL1 and/or BBAP protein. For example, nucleotidesubstitutions leading to amino acid substitutions at “non-essential”amino acid residues can be made in the sequence of SEQ ID NO: 1, 3, 5,7, 9, or 11, or fragment thereof. A “non-essential” amino acid residueis a residue that can be altered from the wild-type sequence of BAL1and/or BBAP (e.g., the sequence of SEQ ID NO: 2, 4, 6, 8, 10, or 12, orfragment thereof) without altering the activity of BAL1 and/or BBAP,whereas an “essential” amino acid residue is required for BAL1 and/orBBAP activity. Other amino acid residues, however, (e.g., those that arenot conserved or only semi-conserved between mouse and human) may not beessential for activity and thus are likely to be amenable to alterationwithout altering BAL1 and/or BBAP activity.

Accordingly, another aspect of the invention pertains to nucleic acidmolecules encoding BAL1 and/or BBAP proteins that contain changes inamino acid residues that are not essential for BAL1 and/or BBAPactivity. Such BAL1 and/or BBAP proteins differ in amino acid sequencefrom SEQ ID NO: 2, 4, 6, 8, 10, or 12, or fragment thereof, yet retainat least one of the BAL1 and/or BBAP activities described herein. In oneembodiment, the isolated nucleic acid molecule comprises a nucleotidesequence encoding a protein, wherein the protein lacks one or more BAL1and/or BBAP domains (e.g., BAL1-binding, BBAP-binding, BBAPdimerization, macro1, macro 2, PARP, and/or RING domains).

The term “sequence identity or homology” refers to the sequencesimilarity between two polypeptide molecules or between two nucleic acidmolecules. When a position in both of the two compared sequences isoccupied by the same base or amino acid monomer subunit, e.g., if aposition in each of two DNA molecules is occupied by adenine, then themolecules are homologous or sequence identical at that position. Thepercent of homology or sequence identity between two sequences is afunction of the number of matching or homologous identical positionsshared by the two sequences divided by the number of positions compared×100. For example, if 6 of 10, of the positions in two sequences are thesame then the two sequences are 60% homologous or have 60% sequenceidentity. By way of example, the DNA sequences ATTGCC and TATGGC share50% homology or sequence identity. Generally, a comparison is made whentwo sequences are aligned to give maximum homology. Unless otherwisespecified “loop out regions”, e.g., those arising from, from deletionsor insertions in one of the sequences are counted as mismatches.

The comparison of sequences and determination of percent homologybetween two sequences can be accomplished using a mathematicalalgorithm. Preferably, the alignment can be performed using the ClustalMethod. Multiple alignment parameters include GAP Penalty=10, Gap LengthPenalty=10. For DNA alignments, the pairwise alignment parameters can beHtuple=2, Gap penalty=5, Window=4, and Diagonal saved=4. For proteinalignments, the pairwise alignment parameters can be Ktuple=1, Gappenalty=3, Window=5, and Diagonals Saved=5.

In a preferred embodiment, the percent identity between two amino acidsequences is determined using the Needleman and Wunsch (J. Mol. Biol.(48):444-453 (1970)) algorithm which has been incorporated into the GAPprogram in the GCG software package (available online), using either aBlossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12,10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yetanother preferred embodiment, the percent identity between twonucleotide sequences is determined using the GAP program in the GCGsoftware package (available online), using a NWSgapdna.CMP matrix and agap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4,5, or 6. In another embodiment, the percent identity between two aminoacid or nucleotide sequences is determined using the algorithm of E.Meyers and W. Miller (CABIOS, 4:11-17 (1989)) which has beenincorporated into the ALIGN program (version 2.0) (available online),using a PAM120 weight residue table, a gap length penalty of 12 and agap penalty of 4.

An isolated nucleic acid molecule encoding a BAL1 and/or BBAP proteinhomologous to the protein of SEQ ID NO: 2, 4, 6, 8, 10, or 12, orfragment thereof, can be created by introducing one or more nucleotidesubstitutions, additions or deletions into the nucleotide sequence ofSEQ ID NO: 1, 3, 5, 7, 9, or 11, or fragment thereof, or a homologousnucleotide sequence such that one or more amino acid substitutions,additions or deletions are introduced into the encoded protein.Mutations can be introduced into SEQ ID NO: 1, 3, 5, 7, 9, or 11, orfragment thereof, or the homologous nucleotide sequence by standardtechniques, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Preferably, conservative amino acid substitutions are madeat one or more predicted non-essential amino acid residues. A“conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), bet217-420 ranched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, apredicted nonessential amino acid residue in BAL1 and/or BBAP ispreferably replaced with another amino acid residue from the same sidechain family. Alternatively, in another embodiment, mutations can beintroduced randomly along all or part of a BAL1 and/or BBAP codingsequence, such as by saturation mutagenesis, and the resultant mutantscan be screened for a BAL1 and/or BBAP activity described herein toidentify mutants that retain BAL1 and/or BBAP activity. Followingmutagenesis of SEQ ID NO: 1, 3, 5, 7, 9, or 11, or fragment thereof, theencoded protein can be expressed recombinantly (as described herein) andthe activity of the protein can be determined using, for example, assaysdescribed herein.

BAL1 and/or BBAP levels may be assessed by any of a wide variety of wellknown methods for detecting expression of a transcribed molecule orprotein. Non-limiting examples of such methods include immunologicalmethods for detection of proteins, protein purification methods, proteinfunction or activity assays, nucleic acid hybridization methods, nucleicacid reverse transcription methods, and nucleic acid amplificationmethods.

In preferred embodiments, BAL1 and/or BBAP levels are ascertained bymeasuring gene transcript (e.g., mRNA), by a measure of the quantity oftranslated protein, or by a measure of gene product activity. Expressionlevels can be monitored in a variety of ways, including by detectingmRNA levels, protein levels, or protein activity, any of which can bemeasured using standard techniques. Detection can involve quantificationof the level of gene expression (e.g., genomic DNA, cDNA, mRNA, protein,or enzyme activity), or, alternatively, can be a qualitative assessmentof the level of gene expression, in particular in comparison with acontrol level. The type of level being detected will be clear from thecontext.

In a particular embodiment, the BAL1 and/or BBAP mRNA expression levelcan be determined both by in situ and by in vitro formats in abiological sample using methods known in the art. The term “biologicalsample” is intended to include tissues, cells, biological fluids andisolates thereof, isolated from a subject, as well as tissues, cells andfluids present within a subject. Many expression detection methods useisolated RNA. For in vitro methods, any RNA isolation technique thatdoes not select against the isolation of mRNA can be utilized for thepurification of RNA from cells (see, e.g., Ausubel et al., ed., CurrentProtocols in Molecular Biology, John Wiley & Sons, New York 1987-1999).Additionally, large numbers of tissue samples can readily be processedusing techniques well known to those of skill in the art, such as, forexample, the single-step RNA isolation process of Chomczynski (1989,U.S. Pat. No. 4,843,155).

The isolated mRNA can be used in hybridization or amplification assaysthat include, but are not limited to, Southern or Northern analyses,polymerase chain reaction analyses and probe arrays. One preferreddiagnostic method for the detection of mRNA levels involves contactingthe isolated mRNA with a nucleic acid molecule (probe) that canhybridize to the mRNA encoded by the gene being detected. The nucleicacid probe can be, for example, a full-length cDNA, or a portionthereof, such as an oligonucleotide of at least 7, 15, 30, 50, 100, 250or 500 nucleotides in length and sufficient to specifically hybridizeunder stringent conditions to a mRNA or genomic DNA encoding BAL1 and/orBBAP. Other suitable probes for use in the diagnostic assays of theinvention are described herein. Hybridization of an mRNA with the probeindicates that BAL1 and/or BBAP is being expressed.

In one format, the mRNA is immobilized on a solid surface and contactedwith a probe, for example by running the isolated mRNA on an agarose geland transferring the mRNA from the gel to a membrane, such asnitrocellulose. In an alternative format, the probe(s) are immobilizedon a solid surface and the mRNA is contacted with the probe(s), forexample, in a gene chip array, e.g., an Affymetrix™ gene chip array. Askilled artisan can readily adapt known mRNA detection methods for usein detecting the level of the BAL1 and/or BBAP mRNA expression levels.

An alternative method for determining the BAL1 and/or BBAP mRNAexpression level in a sample involves the process of nucleic acidamplification, e.g., by rtPCR (the experimental embodiment set forth inMullis, 1987, U.S. Pat. No. 4,683,202), ligase chain reaction (Barany,1991, Proc. Natl. Acad. Sci. USA, 88:189-193), self sustained sequencereplication (Guatelli et al., 1990, Proc. Natl. Acad. Sci. USA87:1874-1878), transcriptional amplification system (Kwoh et al., 1989,Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi etal., 1988, Bio/Technology 6:1197), rolling circle replication (Lizardiet al., U.S. Pat. No. 5,854,033) or any other nucleic acid amplificationmethod, followed by the detection of the amplified molecules usingtechniques well-known to those of skill in the art. These detectionschemes are especially useful for the detection of nucleic acidmolecules if such molecules are present in very low numbers. As usedherein, amplification primers are defined as being a pair of nucleicacid molecules that can anneal to 5′ or 3′ regions of a gene (plus andminus strands, respectively, or vice-versa) and contain a short regionin between. In general, amplification primers are from about 10 to 30nucleotides in length and flank a region from about 50 to 200nucleotides in length. Under appropriate conditions and with appropriatereagents, such primers permit the amplification of a nucleic acidmolecule comprising the nucleotide sequence flanked by the primers.

For in situ methods, mRNA does not need to be isolated from the cellsprior to detection. In such methods, a cell or tissue sample isprepared/processed using known histological methods. The sample is thenimmobilized on a support, typically a glass slide, and then contactedwith a probe that can hybridize to the BAL1 and/or BBAP mRNA.

As an alternative to making determinations based on the absolute BAL1and/or BBAP expression level, determinations may be based on thenormalized BAL1 and/or BBAP expression level. Expression levels arenormalized by correcting the absolute BAL1 and/or BBAP expression levelby comparing its expression to the expression of a non-BAL1 and/or BBAPgene, e.g., a housekeeping gene that is constitutively expressed.Suitable genes for normalization include housekeeping genes such as theactin gene, or epithelial cell-specific genes. This normalization allowsthe comparison of the expression level in one sample, e.g., a subjectsample, to another sample, e.g., a normal sample, or between samplesfrom different sources.

The level or activity of a BAL1 and/or BBAP protein can also be detectedand/or quantified by detecting or quantifying the expressed polypeptide.The BAL1 and/or BBAP polypeptide can be detected and quantified by anyof a number of means well known to those of skill in the art. These mayinclude analytic biochemical methods such as electrophoresis, capillaryelectrophoresis, high performance liquid chromatography (HPLC), thinlayer chromatography (TLC), hyperdiffusion chromatography, and the like,or various immunological methods such as fluid or gel precipitinreactions, immunodiffusion (single or double), immunoelectrophoresis,radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs),immunofluorescent assays, Western blotting, and the like. A skilledartisan can readily adapt known protein/antibody detection methods foruse in determining whether cells express BAL1 and/or BBAP.

In addition to the nucleic acid molecules encoding BAL1 and/or BBAPproteins described above, another aspect of the invention pertains toisolated nucleic acid molecules which are antisense thereto. An“antisense” nucleic acid comprises a nucleotide sequence which iscomplementary to a “sense” nucleic acid encoding a protein, i.e.,complementary to the coding strand of a double-stranded cDNA molecule orcomplementary to an mRNA sequence. Accordingly, an antisense nucleicacid can hydrogen bond to a sense nucleic acid. The antisense nucleicacid can be complementary to an entire BAL1 and/or BBAP coding strand,or to only a portion thereof. In one embodiment, an antisense nucleicacid molecule is antisense to a “coding region” of the coding strand ofa nucleotide sequence encoding BAL1 and/or BBAP. The term “codingregion” refers to the region of the nucleotide sequence comprisingcodons which are translated into amino acid residues. In anotherembodiment, the antisense nucleic acid molecule is antisense to a“noncoding region” of the coding strand of a nucleotide sequenceencoding BAL1 and/or BBAP. The term “noncoding region” refers to 5′ and3′ sequences which flank the coding region that are not translated intoamino acids (i.e., also referred to as 5′ and 3′ untranslated regions).

In some embodiments, BAL1 and/or BBAP expression can be reduced usingnucleic acid compositions described herein. For example, an “RNAinterfering agent,” as used herein, is defined as any agent whichinterferes with or inhibits expression of a target gene, e.g., BAL1and/or BBAP, by RNA interference (RNAi). Such RNA interfering agentsinclude, but are not limited to, nucleic acid molecules including RNAmolecules which are homologous to the target gene or a fragment thereof,short interfering RNA (siRNA), and small molecules which interfere withor inhibit expression of a target gene by RNA interference (RNAi).

“RNA interference (RNAi)” is an evolutionally conserved process wherebythe expression or introduction of RNA of a sequence that is identical orhighly similar to a target gene results in the sequence specificdegradation or specific post-transcriptional gene silencing (PTGS) ofmessenger RNA (mRNA) transcribed from that targeted gene (see Coburn, G.and Cullen, B. (2002) J. of Virology 76(18):9225), thereby inhibitingexpression of the target gene. In one embodiment, the RNA is doublestranded RNA (dsRNA). This process has been described in plants,invertebrates, and mammalian cells. In nature, RNAi is initiated by thedsRNA-specific endonuclease Dicer, which promotes processive cleavage oflong dsRNA into double-stranded fragments termed siRNAs. siRNAs areincorporated into a protein complex that recognizes and cleaves targetmRNAs. RNAi can also be initiated by introducing nucleic acid molecules,e.g., synthetic siRNAs or RNA interfering agents, to inhibit or silencethe expression of target genes. As used herein, “inhibition of targetgene expression” or “inhibition of marker gene expression” includes anydecrease in expression or protein activity or level of the target geneor protein encoded by the target gene. The decrease may be of at least30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or more as compared to theexpression of a target gene or the activity or level of the proteinencoded by a target gene which has not been targeted by an RNAinterfering agent.

“Short interfering RNA” (siRNA), also referred to herein as “smallinterfering RNA” is defined as an agent which functions to inhibitexpression of a target gene, e.g., by RNAi. An siRNA may be chemicallysynthesized, may be produced by in vitro transcription, or may beproduced within a host cell. In one embodiment, siRNA is a doublestranded RNA (dsRNA) molecule of about 15 to about 40 nucleotides inlength, preferably about 15 to about 28 nucleotides, more preferablyabout 19 to about 25 nucleotides in length, and more preferably about19, 20, 21, or 22 nucleotides in length, and may contain a 3′ and/or 5′overhang on each strand having a length of about 0, 1, 2, 3, 4, or 5nucleotides. The length of the overhang is independent between the twostrands, i.e., the length of the over hang on one strand is notdependent on the length of the overhang on the second strand. Preferablythe siRNA is capable of promoting RNA interference through degradationor specific post-transcriptional gene silencing (PTGS) of the targetmessenger RNA (mRNA).

In another embodiment, an siRNA is a small hairpin (also called stemloop) RNA (shRNA). In one embodiment, these shRNAs are composed of ashort (e.g., 19-25 nucleotide) antisense strand, followed by a 5-9nucleotide loop, and the analogous sense strand. Alternatively, thesense strand may precede the nucleotide loop structure and the antisensestrand may follow. These shRNAs may be contained in plasmids,retroviruses, and lentiviruses and expressed from, for example, the polIII U6 promoter, or another promoter (see, e.g., Stewart, et al. (2003)RNA April; 9(4):493-501 incorporated be reference herein).

RNA interfering agents, e.g., siRNA molecules, may be administered to asubject having or at risk for a condition described herein mediated byBAL1 and/or BBAP, to inhibit expression of BAL1 and/or BBAP to therebytreat, prevent, or inhibit the condition in the subject.

II. Recombinant Expression Vectors and Host Cells

Another aspect of the invention pertains to the use of vectors,preferably expression vectors, containing a nucleic acid encoding BAL1and/or BBAP (or a portion or complex thereof). As used herein, the term“vector” refers to a nucleic acid molecule capable of transportinganother nucleic acid to which it has been linked. One type of vector isa “plasmid”, which refers to a circular double stranded DNA loop intowhich additional DNA segments can be ligated. Another type of vector isa viral vector, wherein additional DNA segments can be ligated into theviral genome. Certain vectors are capable of autonomous replication in ahost cell into which they are introduced (e.g., bacterial vectors havinga bacterial origin of replication and episomal mammalian vectors). Othervectors (e.g., non-episomal mammalian vectors) are integrated into thegenome of a host cell upon introduction into the host cell, and therebyare replicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “expressionvectors.” In general, expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. In the presentspecification, “plasmid” and “vector” can be used interchangeably as theplasmid is the most commonly used form of vector. However, the inventionis intended to include such other forms of expression vectors, such asviral vectors (e.g., replication defective retroviruses, adenovirusesand adeno-associated viruses), which serve equivalent functions. In oneembodiment, adenoviral vectors comprising a BAL1 and/or BBAP nucleicacid molecule are used.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell, which means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, which is operatively linked to thenucleic acid sequence to be expressed. Within a recombinant expressionvector, “operably linked” is intended to mean that the nucleotidesequence of interest is linked to the regulatory sequence(s) in a mannerwhich allows for expression of the nucleotide sequence (e.g., in an invitro transcription/translation system or in a host cell when the vectoris introduced into the host cell). The term “regulatory sequence” isintended to include promoters, enhancers and other expression controlelements (e.g., polyadenylation signals). Such regulatory sequences aredescribed, for example, in Goeddel; Gene Expression Technology: Methodsin Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatorysequences include those which direct constitutive expression of anucleotide sequence in many types of host cell and those which directexpression of the nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). It will be appreciated by thoseskilled in the art that the design of the expression vector can dependon such factors as the choice of the host cell to be transformed, thelevel of expression of protein desired, etc. The expression vectors ofthe invention can be introduced into host cells to thereby produceproteins or peptides, including fusion proteins or peptides, encoded bynucleic acids as described herein.

The recombinant expression vectors of the invention can be designed forexpression of BAL1 and/or BBAP in prokaryotic or eukaryotic cells. Forexample, BAL1 and/or BBAP can be expressed in bacterial cells such as E.coli, insect cells (using baculovirus expression vectors) yeast cells ormammalian cells. Suitable host cells are discussed further in Goeddel,Gene Expression Technology: Methods in Enzymology 185, Academic Press,San Diego, Calif. (1990). Alternatively, the recombinant expressionvector can be transcribed and translated in vitro, for example using T7promoter regulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out in E.coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: 1) to increase expression ofrecombinant protein; 2) to increase the solubility of the recombinantprotein; and 3) to aid in the purification of the recombinant protein byacting as a ligand in affinity purification. Often, in fusion expressionvectors, a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent topurification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith, D. B. and Johnson, K. S. (1988) Gene 67:31-40), pMAL (New EnglandBiolabs, Beverly, Mass.) and pRITS (Pharmacia, Piscataway, N.J.) whichfuse glutathione S-transferase (GST), maltose E binding protein, orprotein A, respectively, to the target recombinant protein. In oneembodiment, the coding sequence of the BAL1 and/o BBAP is cloned into apGEX expression vector to create a vector encoding a fusion proteincomprising, from the N-terminus to the C-terminus, and/or GST-thrombincleavage site-BAL1 and/oBBAP. The fusion protein can be purified byaffinity chromatography using glutathione-agarose resin. RecombinantBAL1 and/or BBAP unfused to GST can be recovered by cleavage of thefusion protein with thrombin.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amann et al., (1988) Gene 69:301-315) and pET 11d (Studieret al., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990) 60-89). Target gene expression from thepTrc vector relies on host RNA polymerase transcription from a hybridtrp-lac fusion promoter. Target gene expression from the pET 11d vectorrelies on transcription from a T7 gn10-lac fusion promoter mediated by acoexpressed viral RNA polymerase (T7 gn1). This viral polymerase issupplied by host strains BL21(DE3) or HMS174(DE3) from a resident λprophage harboring a T7 gn1 gene under the transcriptional control ofthe lacUV 5 promoter.

One strategy to maximize recombinant protein expression in E. coli is toexpress the protein in a host bacteria with an impaired capacity toproteolytically cleave the recombinant protein (Gottesman, S., GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990) 119-128). Another strategy is to alter the nucleicacid sequence of the nucleic acid to be inserted into an expressionvector so that the individual codons for each amino acid are thosepreferentially utilized in E. coli (Wada et al. (1992) Nucleic AcidsRes. 20:2111-2118). Such alteration of nucleic acid sequences of theinvention can be carried out by standard DNA synthesis techniques.

In another embodiment, the BAL1 and/or BBAP expression vector is a yeastexpression vector. Examples of vectors for expression in yeast S.cerivisae include pYepSec1 (Baldari, et al., (1987) EMBO J. 6:229-234),pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz etal., (1987) Gene 54:113-123), and pYES2 (Invitrogen Corporation, SanDiego, Calif.).

Alternatively, BAL1 and/or BBAP can be expressed in insect cells usingbaculovirus expression vectors. Baculovirus vectors available forexpression of proteins in cultured insect cells (e.g., Sf 9 cells)include the pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165)and the pVL series (Lucklow and Summers (1989) Virology 170:31-39).

In yet another embodiment, a nucleic acid of the invention is expressedin mammalian cells using a mammalian expression vector. Examples ofmammalian expression vectors include pCDM8 (Seed, B. (1987) Nature329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187-195). When usedin mammalian cells, the expression vector's control functions are oftenprovided by viral regulatory elements. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, cytomegalovirus andSimian Virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J.,Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual.2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert et al.(1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame andEaton (1988) Adv. Immunol. 43:235-275), in particular promoters of Tcell receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) andimmunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen andBaltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci.USA 86:5473-5477), pancreas-specific promoters (Edlund et al. (1985)Science 230:912-916), and mammary gland-specific promoters (e.g., milkwhey promoter; U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, for example the murine hox promoters (Kessel and Gruss(1990) Science 249:374-379) and the α-fetoprotein promoter (Campes andTilghman (1989) Genes Dev. 3:537-546).

The invention further provides a recombinant expression vectorcomprising a nucleic acid molecule of the invention cloned into theexpression vector in an antisense orientation. That is, the DNA moleculeis operatively linked to a regulatory sequence in a manner which allowsfor expression (by transcription of the DNA molecule) of an RNA moleculewhich is antisense to BAL1 and/or BBAP mRNA. Regulatory sequencesoperatively linked to a nucleic acid cloned in the antisense orientationcan be chosen which direct the continuous expression of the antisenseRNA molecule in a variety of cell types, for instance viral promotersand/or enhancers, or regulatory sequences can be chosen which directconstitutive, tissue specific or cell type specific expression ofantisense RNA. The antisense expression vector can be in the form of arecombinant plasmid, phagemid or attenuated virus in which antisensenucleic acids are produced under the control of a high efficiencyregulatory region, the activity of which can be determined by the celltype into which the vector is introduced.

Another aspect of the invention pertains to host cells into which arecombinant expression vector of the invention has been introduced. Theterms “host cell” and “recombinant host cell” are used interchangeablyherein. It is understood that such terms refer not only to theparticular subject cell but to the progeny or potential progeny of sucha cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

A host cell can be any prokaryotic or eukaryotic cell. For example, BAL1and/or BBAP protein can be expressed in bacterial cells such as E. coli,insect cells, yeast or mammalian cells (such as Fao hepatoma cells,primary hepatocytes, Chinese hamster ovary cells (CHO) or COS cells).Other suitable host cells are known to those skilled in the art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid (e.g., DNA) into a host cell, including calcium phosphate orcalcium chloride co-precipitation, DEAE-dextran-mediated transfection,lipofection, or electroporation. Suitable methods for transforming ortransfecting host cells can be found in Sambrook et al. (MolecularCloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989),and other laboratory manuals.

A cell culture includes host cells, media and other byproducts. Suitablemedia for cell culture are well known in the art. A BAL1 and/or BBAPpolypeptide or fragment thereof, may be secreted and isolated from amixture of cells and medium containing the polypeptide. Alternatively, aBAL1 and/or BBAP polypeptide or fragment thereof, may be retainedcytoplasmically and the cells harvested, lysed and the protein orprotein complex isolated. A BAL1 and/or BBAP polypeptide or fragmentthereof, may be isolated from cell culture medium, host cells, or bothusing techniques known in the art for purifying proteins, includingion-exchange chromatography, gel filtration chromatography,ultrafiltration, electrophoresis, and inmmunoaffinity purification withantibodies specific for particular epitopes of BAL1 and/or BBAP or afragment thereof. In other embodiments, heterologous tags can be usedfor purification purposes (e.g., epitope tags and FC fusion tags),according to standards methods known in the art.

Thus, a nucleotide sequence encoding all or a selected portion of a BAL1and/or BBAP polypeptide may be used to produce a recombinant form of theprotein via microbial or eukaryotic cellular processes. Ligating thesequence into a polynucleotide construct, such as an expression vector,and transforming or transfecting into hosts, either eukaryotic (yeast,avian, insect or mammalian) or prokaryotic (bacterial cells), arestandard procedures. Similar procedures, or modifications thereof, maybe employed to prepare recombinant BAL1 and/or BBAP polypeptides, orfragments thereof, by microbial means or tissue-culture technology inaccord with the subject invention.

In another variation, protein production may be achieved using in vitrotranslation systems. In vitro translation systems are, generally, atranslation system which is a cell-free extract containing at least theminimum elements necessary for translation of an RNA molecule into aprotein. An in vitro translation system typically comprises at leastribosomes, tRNAs, initiator methionyl-tRNAMet, proteins or complexesinvolved in translation, e.g., eIF2, eIF3, the cap-binding (CB) complex,comprising the cap-binding protein (CBP) and eukaryotic initiationfactor 4F (eIF4F). A variety of in vitro translation systems are wellknown in the art and include commercially available kits. Examples of invitro translation systems include eukaryotic lysates, such as rabbitreticulocyte lysates, rabbit oocyte lysates, human cell lysates, insectcell lysates and wheat germ extracts. Lysates are commercially availablefrom manufacturers such as Promega Corp., Madison, Wis.; Stratagene, LaJolla, Calif.; Amersham, Arlington Heights, Ill.; and GIBCO/BRL, GrandIsland, N.Y. In vitro translation systems typically comprisemacromolecules, such as enzymes, translation, initiation and elongationfactors, chemical reagents, and ribosomes. In addition, an in vitrotranscription system may be used. Such systems typically comprise atleast an RNA polymerase holoenzyme, ribonucleotides and any necessarytranscription initiation, elongation and termination factors. In vitrotranscription and translation may be coupled in a one-pot reaction toproduce proteins from one or more isolated DNAs.

In certain embodiments, the BAL1 and/or BBAP polypeptide, or fragmentthereof, may be synthesized chemically, ribosomally in a cell freesystem, or ribosomally within a cell. Chemical synthesis may be carriedout using a variety of art recognized methods, including stepwise solidphase synthesis, semi-synthesis through the conformationally-assistedre-ligation of peptide fragments, enzymatic ligation of cloned orsynthetic peptide segments, and chemical ligation. Native chemicalligation employs a chemoselective reaction of two unprotected peptidesegments to produce a transient thioester-linked intermediate. Thetransient thioester-linked intermediate then spontaneously undergoes arearrangement to provide the full length ligation product having anative peptide bond at the ligation site. Full-length ligation productsare chemically identical to proteins produced by cell free synthesis.Full length ligation products may be refolded and/or oxidized, asallowed, to form native disulfide-containing protein molecules. (seee.g., U.S. Pat. Nos. 6,184,344 and 6,174,530; and T. W. Muir et al.,Curr. Opin. Biotech. (1993): vol. 4, p 420; M. Miller, et al., Science(1989): vol. 246, p 1149; A. Wlodawer, et al., Science (1989): vol. 245,p 616; L. H. Huang, et al., Biochemistry (1991): vol. 30, p 7402; M.Sclmolzer, et al., Int. J. Pept. Prot. Res. (1992): vol. 40, p 180-193;K. Rajarathnam, et al., Science (1994): vol. 264, p 90; R. E. Offord,“Chemical Approaches to Protein Engineering”, in Protein Design and theDevelopment of New therapeutics and Vaccines, J. B. Hook, G. Poste,Eds., (Plenum Press, New York, 1990) pp. 253-282; C. J. A. Wallace, etal., J. Biol. Chem. (1992): vol. 267, p 3852; L. Abrahmsen, et al.,Biochemistry (1991): vol. 30, p 4151; T. K. Chang, et al., Proc. Natl.Acad. Sci. USA (1994) 91: 12544-12548; M. Schnlzer, et al., Science(1992): vol., 3256, p 221; and K. Akaji, et al., Chem. Pharm. Bull.(Tokyo) (1985) 33: 184).

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest.Preferred selectable markers include those which confer resistance todrugs, such as G418, hygromycin and methotrexate. Nucleic acid encodinga selectable marker can be introduced into a host cell on the samevector as that encoding BAL1 and/or BBAP or can be introduced on aseparate vector. Cells stably transfected with the introduced nucleicacid can be identified by drug selection (e.g., cells that haveincorporated the selectable marker gene will survive, while the othercells die).

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce (i.e., express) BAL1 and/or BBAPprotein. Accordingly, the invention further provides methods forproducing BAL1 and/or BBAP protein using the host cells of theinvention. In one embodiment, the method comprises culturing the hostcell of invention (into which a recombinant expression vector encodingBAL1 and/or BBAP has been introduced) in a suitable medium until BAL1and/or BBAP is produced. In another embodiment, the method furthercomprises isolating BAL1 and/or BBAP from the medium or the host cell.

The host cells of the invention can also be used to produce nonhumantransgenic animals. The nonhuman transgenic animals can be used inscreening assays designed to identify agents or compounds, e.g., drugs,pharmaceuticals, etc., which are capable of ameliorating detrimentalsymptoms of selected disorders such as glucose homeostasis disorders,weight disorders or disorders associated with insufficient insulinactivity. For example, in one embodiment, a host cell of the inventionis a fertilized oocyte or an embryonic stem cell into which BAL1 and/orBBAP encoding sequences, or fragments thereof, have been introduced.Such host cells can then be used to create non-human transgenic animalsin which exogenous BAL1 and/or BBAP sequences have been introduced intotheir genome or homologous recombinant animals in which endogenous BAL1and/or BBAP sequences have been altered. Such animals are useful forstudying the function and/or activity of BAL1 and/or BBAP, or fragmentsthereof, and for identifying and/or evaluating modulators of BAL1 and/orBBAP activity. As used herein, a “transgenic animal” is a nonhumananimal, preferably a mammal, more preferably a rodent such as a rat ormouse, in which one or more of the cells of the animal includes atransgene. Other examples of transgenic animals include nonhumanprimates, sheep, dogs, cows, goats, chickens, amphibians, etc. Atransgene is exogenous DNA which is integrated into the genome of a cellfrom which a transgenic animal develops and which remains in the genomeof the mature animal, thereby directing the expression of an encodedgene product in one or more cell types or tissues of the transgenicanimal. As used herein, a “homologous recombinant animal” is a nonhumananimal, preferably a mammal, more preferably a mouse, in which anendogenous BAL1 and/or BBAP gene has been altered by homologousrecombination between the endogenous gene and an exogenous DNA moleculeintroduced into a cell of the animal, e.g., an embryonic cell of theanimal, prior to development of the animal.

A transgenic animal of the invention can be created by introducingnucleic acids encoding BAL1 and/or BBAP, or a fragment thereof, into themale pronuclei of a fertilized oocyte, e.g., by microinjection,retroviral infection, and allowing the oocyte to develop in apseudopregnant female foster animal. A human BAL1 and/or BBAP cDNAsequence can be introduced as a transgene into the genome of a nonhumananimal. Alternatively, a nonhuman homologue of the human BAL1 and/orBBAP gene can be used as a transgene. Intronic sequences andpolyadenylation signals can also be included in the transgene toincrease the efficiency of expression of the transgene. Atissue-specific regulatory sequence(s) can be operably linked to theBAL1 and/or BBAP transgene to direct expression of BAL1 and/or BBAPprotein to particular cells. Methods for generating transgenic animalsvia embryo manipulation and microinjection, particularly animals such asmice, have become conventional in the art and are described, forexample, in U.S. Pat. Nos. 4,736,866 and 4,870,009, both by Leder etal., U.S. Pat. No. 4,873,191 by Wagner et al. and in Hogan, B.,Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1986). Similar methods are used for productionof other transgenic animals. A transgenic founder animal can beidentified based upon the presence of the BAL1 and/or BBAP transgene inits genome and/or expression of BAL1 and/or BBAP mRNA in tissues orcells of the animals. A transgenic founder animal can then be used tobreed additional animals carrying the transgene. Moreover, transgenicanimals carrying a transgene encoding BAL1 and/or BBAP can further bebred to other transgenic animals carrying other transgenes.

To create a homologous recombinant animal, a vector is prepared whichcontains at least a portion of a BAL1 and/or BBAP gene into which adeletion, addition or substitution has been introduced to thereby alter,e.g., functionally disrupt, the BAL1 and/or BBAP gene. The BAL1 and/orBBAP gene can be a human gene, but more preferably, is a nonhumanhomologue of a human BAL1 and/or BBAP gene. For example, a mouse BAL1and/or BBAP gene can be used to construct a homologous recombinationvector suitable for altering an endogenous BAL1 and/or BBAP gene,respectively, in the mouse genome. In a preferred embodiment, the vectoris designed such that, upon homologous recombination, the endogenousBAL1 and/or BBAP gene is functionally disrupted in some manner (e.g., nolonger encodes a functional protein, no longer encodes one or morefunctional domain(s), etc.; also referred to as a “knock out” vector).Alternatively, the vector can be designed such that, upon homologousrecombination, the endogenous BAL1 and/or BBAP gene is mutated orotherwise altered but still encodes functional protein (e.g., theupstream regulatory region can be altered to thereby alter theexpression of the endogenous BAL1 and/or BBAP protein). In thehomologous recombination vector, the altered portion of the BAL1 and/orBBAP gene is flanked at its 5′ and 3′ ends by additional nucleic acid ofthe BAL1 and/or BBAP gene to allow for homologous recombination to occurbetween the exogenous BAL1 and/or BBAP gene carried by the vector and anendogenous BAL1 and/or BBAP gene in an embryonic stem cell. Theadditional flanking BAL1 and/or BBAP nucleic acid is of sufficientlength for successful homologous recombination with the endogenous gene.Typically, several kilobases of flanking DNA (both at the 5′ and 3′ends) are included in the vector (see e.g., Thomas, K. R. and Capecchi,M. R. (1987) Cell 51:503 for a description of homologous recombinationvectors). The vector is introduced into an embryonic stem cell line(e.g., by electroporation) and cells in which the introduced BAL1 and/orBBAP gene has homologously recombined with the endogenous BAL1 and/orBBAP gene are selected (see e.g., Li, E. et al. (1992) Cell 69:915). Theselected cells are then injected into a blastocyst of an animal (e.g., amouse) to form aggregation chimeras (see e.g., Bradley, A. inTeratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J.Robertson, ed. (IRL, Oxford, 1987) pp. 113-152). A chimeric embryo canthen be implanted into a suitable pseudopregnant female foster animaland the embryo brought to term. Progeny harboring the homologouslyrecombined DNA in their germ cells can be used to breed animals in whichall cells of the animal contain the homologously recombined DNA bygermline transmission of the transgene. Methods for constructinghomologous recombination vectors and homologous recombinant animals aredescribed further in Bradley, A. (1991) Current Opinion in Biotechnology2:823-829 and in PCT International Publication Nos. WO 90/11354 by LeMouellec et al.; WO 91/01140 by Smithies et al.; WO 92/0968 by Zijlstraet al.; and WO 93/04169 by Berns et al.

In another embodiment, transgenic nonhuman animals can be produced whichcontain selected systems which allow for regulated expression of thetransgene. One example of such a system is the cre/loxP recombinasesystem of bacteriophage P1. For a description of the cre/loxPrecombinase system, see, e.g., Lakso et al. (1992) Proc. Natl. Acad.Sci. USA 89:6232-6236. Another example of a recombinase system is theFLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al.(1991) Science 251:1351-1355. If a cre/loxP recombinase system is usedto regulate expression of the transgene, animals containing transgenesencoding both the Cre recombinase and a selected protein are required.Such animals can be provided through the construction of “double”transgenic animals, e.g., by mating two transgenic animals, onecontaining a transgene encoding a selected protein and the othercontaining a transgene encoding a recombinase.

Clones of the nonhuman transgenic animals described herein can also beproduced according to the methods described in Wilmut, I. et al. (1997)Nature 385:810-813 and PCT International Publication Nos. WO 97/07668and WO 97/07669. In brief, a cell, e.g., a somatic cell, from thetransgenic animal can be isolated and induced to exit the growth cycleand enter G_(O) phase. The quiescent cell can then be fused, e.g.,through the use of electrical pulses, to an enucleated oocyte from ananimal of the same species from which the quiescent cell is isolated.The reconstructed oocyte is then cultured such that it develops tomorula or blastocyst and then transferred to pseudopregnant femalefoster animal. The offspring borne of this female foster animal will bea clone of the animal from which the cell, e.g., the somatic cell, isisolated.

III. Isolated BAL1 and/or BBAP Polypeptides and Anti-BAL1 and/or BBAPAntibodies

The present invention provides soluble, purified and/or isolated formsof BAL1 and/or BBAP, or fragments thereof. In addition, it is to beunderstood that any and all attributes of the BAL1 and BBAP polypeptidesdescribed herein, such as percentage identities, polypeptide lengths,polypeptide fragments, biological activities, antibodies, etc. can becombined in any order or combination with respect to BAL1, BBAP, and/orBAL1-BBAP complexes.

In one aspect, a BAL1 and/or BBAP polypeptide may comprise a full-lengthBAL1 and/or BBAP amino acid sequence or a full-length BAL1 and/or BBAPamino acid sequence with 1 to about 20 conservative amino acidsubstitutions Amino acid sequence of any BAL1 and/or BBAP polypeptidedescribed herein can also be at least 50, 55, 60, 65, 70, 75, 80, 85,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 99.5% identical to a BAL1and/or BBAP polypeptide sequence of interest, described herein, wellknown in the art, or a fragment thereof. In addition, any BAL1 and/orBBAP polypeptide, or fragment thereof, described herein has modulates(e.g., enhance) one or more of the following biological activities: a)binding to a BAL1 polypeptide or fragment thereof; b) binding to a BBAPpolypeptide or fragment thereof; c) forming a BAL1-BBAP complex; d)inhibiting localization and/or binding of BAL1 and/or BBAP to DNA damagesites; e) inhibiting binding of BAL1 to poly(ADP-ribose) (PAR) chains;f) inhibiting BBAP monoubiquitylation of histones; g) inhibitingBBAP-mediated methylation of histones; h) inhibiting localization and/orbinding to DNA damage sites of at least one polypeptide selected fromthe group consisting of 53 BP1, RAP80, BRCA1, ATM, γH2AX, and MDC1; andi) inhibiting DNA damage responses (DDR). In another aspect, the presentinvention contemplates a composition comprising an isolated BAL1 and/orBBAP polypeptide and less than about 25%, or alternatively 15%, oralternatively 5%, contaminating biological macromolecules orpolypeptides.

The present invention further provides compositions related toproducing, detecting, or characterizing a BAL1 and/or BBAP polypeptide,or fragment thereof, such as nucleic acids, vectors, host cells, and thelike. Such compositions may serve as compounds that modulate a BAL1and/or BBAP polypeptide's expression and/or activity, such as antisensenucleic acids.

In certain embodiments, a BAL1 and/or BBAP polypeptide of the inventionmay be a fusion protein containing a domain which increases itssolubility and bioavailability and/or facilitates its purification,identification, detection, and/or structural characterization. Exemplarydomains, include, for example, glutathione S-transferase (GST), proteinA, protein G, calmodulin-binding peptide, thioredoxin, maltose bindingprotein, HA, myc, poly arginine, poly His, poly His-Asp or FLAG fusionproteins and tags. Additional exemplary domains include domains thatalter protein localization in vivo, such as signal peptides, type IIIsecretion system-targeting peptides, transcytosis domains, nuclearlocalization signals, etc. In various embodiments, a BAL1 and/or BBAPpolypeptide of the invention may comprise one or more heterologousfusions. Polypeptides may contain multiple copies of the same fusiondomain or may contain fusions to two or more different domains. Thefusions may occur in within the polypeptide as an in-frame insertion, atthe N-terminus of the polypeptide, at the C-terminus of the polypeptide,or at both the N- and C-terminus of the polypeptide. It is also withinthe scope of the invention to include linker sequences between apolypeptide of the invention and the fusion domain in order tofacilitate construction of the fusion protein or to optimize proteinexpression or structural constraints of the fusion protein. In anotherembodiment, the polypeptide may be constructed so as to contain proteasecleavage sites between the fusion polypeptide and polypeptide of theinvention in order to remove the tag after protein expression orthereafter. Examples of suitable endoproteases, include, for example,Factor Xa and TEV proteases.

In some embodiments, BAL1 and/or BBAP polypeptides, or fragmentsthereof, are fused to an antibody fragment (e.g., Fc polypeptides).Techniques for preparing these fusion proteins are known, and aredescribed, for example, in WO 99/31241 and in Cosman et. al., 2001Immunity 14:123 133. Fusion to an Fc polypeptide offers the additionaladvantage of facilitating purification by affinity chromatography overProtein A or Protein G columns.

In still another embodiment, a BAL1 and/or BBAP polypeptide may belabeled with a fluorescent label to facilitate their detection,purification, or structural characterization. In an exemplaryembodiment, a BAL1 and/or BBAP polypeptide of the invention may be fusedto a heterologous polypeptide sequence which produces a detectablefluorescent signal, including, for example, green fluorescent protein(GFP), enhanced green fluorescent protein (EGFP), Renilla Reniformisgreen fluorescent protein, GFPmut2, GFPuv4, enhanced yellow fluorescentprotein (EYFP), enhanced cyan fluorescent protein (ECFP), enhanced bluefluorescent protein (EBFP), citrine and red fluorescent protein fromdiscosoma (dsRED).

Another aspect of the invention pertains to the use of isolated BAL1and/or BBAP proteins, and biologically active portions thereof, as wellas peptide fragments suitable for use as immunogens to raise anti-BAL1and/or BBAP antibodies. An “isolated” or “purified” protein orbiologically active portion thereof is substantially free of cellularmaterial when produced by recombinant DNA techniques, or chemicalprecursors or other chemicals when chemically synthesized. The language“substantially free of cellular material” includes preparations of BAL1and/or BBAP protein in which the protein is separated from cellularcomponents of the cells in which it is naturally or recombinantlyproduced. In one embodiment, the language “substantially free ofcellular material” includes preparations of BAL1 and/or BBAP proteinhaving less than about 30% (by dry weight) of non-BAL1 and/or BBAPprotein (also referred to herein as a “contaminating protein”), morepreferably less than about 20% of non-BAL1 and/or BBAP protein, stillmore preferably less than about 10% of non-BAL1 and/or BBAP protein, andmost preferably less than about 5% non-BAL1 and/or BBAP protein. Whenthe BAL1 and/or BBAP protein or biologically active portion thereof isrecombinantly produced, it is also preferably substantially free ofculture medium, i.e., culture medium represents less than about 20%,more preferably less than about 10%, and most preferably less than about5% of the volume of the protein preparation. The language “substantiallyfree of chemical precursors or other chemicals” includes preparations ofBAL1 and/or BBAP protein in which the protein is separated from chemicalprecursors or other chemicals which are involved in the synthesis of theprotein. In one embodiment, the language “substantially free of chemicalprecursors or other chemicals” includes preparations of BAL1 and/or BBAPprotein having less than about 30% (by dry weight) of chemicalprecursors of non-BAL1 and/or BBAP chemicals, more preferably less thanabout 20% chemical precursors of non-BAL1 and/or BBAP chemicals, stillmore preferably less than about 10% chemical precursors of non-BAL1and/or BBAP chemicals, and most preferably less than about 5% chemicalprecursors of non-BAL1 and/or BBAP chemicals. In preferred embodiments,isolated proteins or biologically active portions thereof lackcontaminating proteins from the same animal from which the BAL1 and/orBBAP protein is derived. Typically, such proteins are produced byrecombinant expression of, for example, a human BAL1 and/or BBAP proteinin a nonhuman cell.

In preferred embodiments, the protein or portion thereof comprises anamino acid sequence which is sufficiently homologous to an amino acidsequence of SEQ ID NO: 2, 4, 6, 8, 10, or 12, or fragment thereof, suchthat the protein or portion thereof maintains one or more of thefollowing biological activities or, in complex, modulates (e.g.,enhance) one or more of the following biological activities: a) bindingto a BAL1 polypeptide or fragment thereof; b) binding to a BBAPpolypeptide or fragment thereof; c) forming a BAL1-BBAP complex; d)inhibiting localization and/or binding of BAL1 and/or BBAP to DNA damagesites; e) inhibiting binding of BAL1 to poly(ADP-ribose) (PAR) chains;f) inhibiting BBAP monoubiquitylation of histones; g) inhibitingBBAP-mediated methylation of histones; h) inhibiting localization and/orbinding to DNA damage sites of at least one polypeptide selected fromthe group consisting of 53 BP1, RAP80, BRCA1, ATM, γH2AX, and MDC1; andi) inhibiting DNA damage responses (DDR). The portion of the protein ispreferably a biologically active portion as described herein. In anotherpreferred embodiment, the BAL1 and/or BBAP protein has an amino acidsequence shown in SEQ ID NO: 2, 4, 6, 8, 10, or 12, or fragment thereof,respectively, or an amino acid sequence which is at least about 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more homologous to the amino acid sequence shown in SEQID NO: 2, 4, 6, 8, 10, or 12, or fragment thereof. In yet anotherpreferred embodiment, the BAL1 and/or BBAP protein has an amino acidsequence which is encoded by a nucleotide sequence which hybridizes,e.g., hybridizes under stringent conditions, to the nucleotide sequenceof SEQ ID NO:1, 3, 5, 7, 9, or 11, or fragment thereof, or a nucleotidesequence which is at least about 50%, preferably at least about 60%,more preferably at least about 70%, yet more preferably at least about80%, still more preferably at least about 90%, and most preferably atleast about 95%, 96%, 97%, 98%, 99% or more homologous to the nucleotidesequence shown in SEQ ID NO: 1, 3, 5, 7, 9, or 11, or fragment thereof.The preferred BAL1 and/or BBAP proteins of the present invention alsopreferably possess at least one of the BAL1 and/or BBAP biologicalactivities, or activities associated with the complex, described herein.For example, a preferred BAL1 and/or BBAP protein of the presentinvention includes an amino acid sequence encoded by a nucleotidesequence which hybridizes, e.g., hybridizes under stringent conditions,to the nucleotide sequence of SEQ ID NO:1, 3, 5, 7, 9, or 11, orfragment thereof and which can maintain one or more of the followingbiological activities or, in complex, modulates (e.g., enhance) one ormore of the following biological activities: a) binding to a BAL1polypeptide or fragment thereof; b) binding to a BBAP polypeptide orfragment thereof; c) forming a BAL1-BBAP complex; d) inhibitinglocalization and/or binding of BAL1 and/or BBAP to DNA damage sites; e)inhibiting binding of BAL1 to poly(ADP-ribose) (PAR) chains; f)inhibiting BBAP monoubiquitylation of histones; g) inhibitingBBAP-mediated methylation of histones; h) inhibiting localization and/orbinding to DNA damage sites of at least one polypeptide selected fromthe group consisting of 53 BP1, RAP80, BRCA1, ATM, γH2AX, and MDC1; andi) inhibiting DNA damage responses (DDR).

Biologically active portions of the BAL1 and/or BBAP protein includepeptides comprising amino acid sequences derived from the amino acidsequence of the BAL1 and/or BBAP protein, e.g., the amino acid sequenceshown in SEQ ID NO: 2, 4, 6, 8, 10, or 12, or fragment thereof, or theamino acid sequence of a protein homologous to the BAL1 and/or BBAPprotein, which include fewer amino acids than the full length BAL1and/or BBAP protein or the full length protein which is homologous tothe BAL1 and/or BBAP protein, and exhibit at least one activity of theBAL1 and/or BBAP protein, or complex thereof. Typically, biologicallyactive portions (peptides, e.g., peptides which are, for example, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more amino acids in length)comprise a domain or motif, e.g., BAL1-binding, BBAP-binding, BBAPdimerization, macro1, macro 2, PARP, and/or RING domains. In a preferredembodiment, the biologically active portion of the protein whichincludes one or more the domains/motifs described herein can modulateDDR, especially in cancer cells. Moreover, other biologically activeportions, in which other regions of the protein are deleted, can beprepared by recombinant techniques and evaluated for one or more of theactivities described herein. Preferably, the biologically activeportions of the BAL1 and/or BBAP protein include one or more selecteddomains/motifs or portions thereof having biological activity. In anexemplary embodiment, a BAL1 and/or BBAP fragment comprises and/orconsists of about amino acids 30-140, 30-140, 73-140, 73-150, 1-140,1-150, or any range in between residues 1 and 150 of SEQ ID NO:2. Inanother embodiment, a BAL1 and/or BBAP fragment consists of a portion ofa full-length BAL1 and/or BBAP fragment of interest that is less than195, 190, 185, 180, 175, 170, 165, 160, 155, 150, 145, 140, 135, 130,125, 120, 115, 110, 105, 100, 95, 90, 85, 80, 75, or 70 amino acids inlength.

BAL1 and/or BBAP proteins can be produced by recombinant DNA techniques.For example, a nucleic acid molecule encoding the protein is cloned intoan expression vector (as described above), the expression vector isintroduced into a host cell (as described above) and the BAL1 and/orBBAP protein is expressed in the host cell. The BAL1 and/or BBAP proteincan then be isolated from the cells by an appropriate purificationscheme using standard protein purification techniques. Alternative torecombinant expression, a BAL1 and/or BBAP protein, polypeptide, orpeptide can be synthesized chemically using standard peptide synthesistechniques. Moreover, native BAL1 and/or BBAP protein can be isolatedfrom cells (e.g., lymphoma cells), for example using an anti-BAL1 and/orBBAP antibody (described further below).

The invention also provides BAL1 and/or BBAP chimeric or fusionproteins. As used herein, a BAL1 and/or BBAP “chimeric protein” or“fusion protein” comprises a BAL1 and/or BBAP polypeptide operativelylinked to a non-BAL1 and/or BBAP polypeptide. A “BAL1 and/or BBAPpolypeptide” refers to a polypeptide having an amino acid sequencecorresponding to BAL1 and/or BBAP, whereas a “non-BAL1 and/or BBAPpolypeptide” refers to a polypeptide having an amino acid sequencecorresponding to a protein which is not substantially homologous to theBAL1 and/or BBAP protein, respectively, e.g., a protein which isdifferent from the BAL1 and/or BBAP protein and which is derived fromthe same or a different organism. Within the fusion protein, the term“operatively linked” is intended to indicate that the BAL1 and/or BBAPpolypeptide and the non-BAL1 and/or BBAP polypeptide are fused in-frameto each other. The non-BAL1 and/or BBAP polypeptide can be fused to theN-terminus or C-terminus of the BAL1 and/or BBAP polypeptide,respectively. For example, in one embodiment the fusion protein is aBAL1 and/or BBAP-GST and/or BAL1 and/or BBAP-Fc fusion protein in whichthe BAL1 and/or BBAP sequences, respectively, are fused to theN-terminus of the GST or Fc sequences. Such fusion proteins canfacilitate the purification, expression, and/or bioavailability ofrecombinant BAL1 and/or BBAP. In another embodiment, the fusion proteinis a BAL1 and/or BBAP protein containing a heterologous signal sequenceat its C-terminus. In certain host cells (e.g., mammalian host cells),expression and/or secretion of BAL1 and/or BBAP can be increased throughuse of a heterologous signal sequence.

Preferably, a BAL1 and/or BBAP chimeric or fusion protein of theinvention is produced by standard recombinant DNA techniques. Forexample, DNA fragments coding for the different polypeptide sequencesare ligated together in-frame in accordance with conventionaltechniques, for example by employing blunt-ended or stagger-endedtermini for ligation, restriction enzyme digestion to provide forappropriate termini, filling-in of cohesive ends as appropriate,alkaline phosphatase treatment to avoid undesirable joining, andenzymatic ligation. In another embodiment, the fusion gene can besynthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers which give rise to complementaryoverhangs between two consecutive gene fragments which can subsequentlybe annealed and reamplified to generate a chimeric gene sequence (see,for example, Current Protocols in Molecular Biology, eds. Ausubel et al.John Wiley & Sons: 1992). Moreover, many expression vectors arecommercially available that already encode a fusion moiety (e.g., a GSTpolypeptide). A BAL1 and/or BBAP-encoding nucleic acid can be clonedinto such an expression vector such that the fusion moiety is linkedin-frame to the BAL1 and/or BBAP protein.

The present invention also pertains to homologues of the BAL1 and/orBBAP proteins which function as either a BAL1 and/or BBAP agonist(mimetic) or a BAL1 and/or BBAP antagonist. In a preferred embodiment,the BAL1 and/or BBAP agonists and antagonists stimulate or inhibit,respectively, a subset of the biological activities of the naturallyoccurring form of the BAL1 and/or BBAP protein. Thus, specificbiological effects can be elicited by treatment with a homologue oflimited function. In one embodiment, treatment of a subject with ahomologue having a subset of the biological activities of the naturallyoccurring form of the protein has fewer side effects in a subjectrelative to treatment with the naturally occurring form of the BAL1and/or BBAP protein. For example, BAL1 and/or BBAP protein fragments canbe used to restrict localization of the BAL1-BBAP complex from thenucleus, thereby inhibiting DDR.

Homologues of the BAL1 and/or BBAP protein can be generated bymutagenesis, e.g., discrete point mutation or truncation of the BAL1and/or BBAP protein. As used herein, the term “homologue” refers to avariant form of the BAL1 and/or BBAP protein which acts as an agonist orantagonist of the activity of the BAL1 and/or BBAP protein. An agonistof the BAL1 and/or BBAP protein can retain substantially the same, or asubset, of the biological activities of the BAL1 and/or BBAP protein. Anantagonist of the BAL1 and/or BBAP protein can inhibit one or more ofthe activities of the naturally occurring form of the BAL1 and/or BBAPprotein, by, for example, competitively binding to a downstream orupstream member of the BAL1 and/or BBAP cascade which includes the BAL1and/or BBAP protein.

In an alternative embodiment, homologues of the BAL1 and/or BBAP proteincan be identified by screening combinatorial libraries of mutants, e.g.,truncation mutants, of the BAL1 and/or BBAP protein for BAL1 and/or BBAPprotein agonist or antagonist activity. In one embodiment, a variegatedlibrary of BAL1 and/or BBAP variants is generated by combinatorialmutagenesis at the nucleic acid level and is encoded by a variegatedgene library. A variegated library of BAL1 and/or BBAP variants can beproduced by, for example, enzymatically ligating a mixture of syntheticoligonucleotides into gene sequences such that a degenerate set ofpotential BAL1 and/or BBAP sequences is expressible as individualpolypeptides, or alternatively, as a set of larger fusion proteins(e.g., for phage display) containing the set of BAL1 and/or BBAPsequences therein. There are a variety of methods which can be used toproduce libraries of potential BAL1 and/or BBAP homologues from adegenerate oligonucleotide sequence. Chemical synthesis of a degenerategene sequence can be performed in an automatic DNA synthesizer, and thesynthetic gene then ligated into an appropriate expression vector. Useof a degenerate set of genes allows for the provision, in one mixture,of all of the sequences encoding the desired set of potential BAL1and/or BBAP sequences. Methods for synthesizing degenerateoligonucleotides are known in the art (see, e.g., Narang, S. A. (1983)Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem. 53:323;Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic AcidRes. 11:477.

In addition, libraries of fragments of the BAL1 and/or BBAP proteincoding can be used to generate a variegated population of BAL1 and/orBBAP fragments for screening and subsequent selection of homologues of aBAL1 and/or BBAP protein. In one embodiment, a library of codingsequence fragments can be generated by treating a double stranded PCRfragment of a BAL1 and/or BBAP coding sequence with a nuclease underconditions wherein nicking occurs only about once per molecule,denaturing the double stranded DNA, renaturing the DNA to form doublestranded DNA which can include sense/antisense pairs from differentnicked products, removing single stranded portions from reformedduplexes by treatment with S1 nuclease, and ligating the resultingfragment library into an expression vector. By this method, anexpression library can be derived which encodes N-terminal, C-terminaland internal fragments of various sizes of the BAL1 and/or BBAP protein.

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation, and forscreening cDNA libraries for gene products having a selected property.Such techniques are adaptable for rapid screening of the gene librariesgenerated by the combinatorial mutagenesis of BAL1 and/or BBAPhomologues. The most widely used techniques, which are amenable to highthrough-put analysis, for screening large gene libraries typicallyinclude cloning the gene library into replicable expression vectors,transforming appropriate cells with the resulting library of vectors,and expressing the combinatorial genes under conditions in whichdetection of a desired activity facilitates isolation of the vectorencoding the gene whose product was detected. Recursive ensemblemutagenesis (REM), a new technique which enhances the frequency offunctional mutants in the libraries, can be used in combination with thescreening assays to identify BAL1 and/or BBAP homologues (Arkin andYouvan (1992) Proc. Natl. Acad. Sci. USA 89:7811-7815; Delagrave et al.(1993) Protein Engineering 6(3):327-331).

In another aspect, an isolated BAL1 and/or BBAP protein, or a fragmentthereof, can be used as an immunogen to generate antibodies that bindBAL1 and/or BBAP, or the complex thereof, using standard techniques forpolyclonal and monoclonal antibody preparation. The full-length BAL1and/or BBAP protein can be used or, alternatively, antigenic peptidefragments of BAL1 and/or BBAP, or peptides in complex, can be used asimmunogens. A BAL1 and/or BBAP immunogen typically is used to prepareantibodies by immunizing a suitable subject, (e.g., rabbit, goat, mouseor other mammal) with the immunogen. An appropriate immunogenicpreparation can contain, for example, recombinantly expressed BAL1and/or BBAP protein or a chemically synthesized BAL1 and/or BBAPpeptide. The preparation can further include an adjuvant, such asFreund's complete or incomplete adjuvant, or similar immunostimulatoryagent Immunization of a suitable subject with an immunogenic BAL1 and/orBBAP preparation induces a polyclonal anti-BAL1 and/or BBAP antibodyresponse.

Accordingly, another aspect of the invention pertains to the use ofanti-BAL1 and/or BBAP antibodies. The term “antibody” as used hereinrefers to immunoglobulin molecules and immunologically active portionsof immunoglobulin molecules, i.e., molecules that contain an antigenbinding site which specifically binds (immunoreacts with) an antigen,such as BAL1 and/or BBAP. Examples of immunologically active portions ofimmunoglobulin molecules include F(ab) and F(ab′)₂ fragments which canbe generated by treating the antibody with an enzyme such as pepsin. Theinvention provides polyclonal and monoclonal antibodies that bind BAL1and/or BBAP. The term “monoclonal antibody” or “monoclonal antibodycomposition”, as used herein, refers to a population of antibodymolecules that contain only one species of an antigen binding sitecapable of immunoreacting with a particular epitope of BAL1 and/or BBAP.A monoclonal antibody composition thus typically displays a singlebinding affinity for a particular BAL1 and/or BBAP protein with which itimmunoreacts.

Polyclonal anti-BAL1 and/or BBAP antibodies can be prepared as describedabove by immunizing a suitable subject with a BAL1 and/or BBAPimmunogen, or fragment thereof. The anti-BAL1 and/or BBAP antibody titerin the immunized subject can be monitored over time by standardtechniques, such as with an enzyme linked immunosorbent assay (ELISA)using immobilized BAL1 and/or BBAP. If desired, the antibody moleculesdirected against BAL1 and/or BBAP can be isolated from the mammal (e.g.,from the blood) and further purified by well known techniques, such asprotein A chromatography to obtain the IgG fraction. At an appropriatetime after immunization, i.e., when the anti-BAL1 and/or BBAP antibodytiters are highest, antibody-producing cells can be obtained from thesubject and used to prepare monoclonal antibodies by standardtechniques, such as the hybridoma technique originally described byKohler and Milstein (1975) Nature 256:495-497) (see also, Brown et al.(1981) J. Immunol. 127:539-46; Brown et al. (1980) J. Biol. Chem.255:4980-83; Yeh et al. (1976) Proc. Natl. Acad. Sci. USA 76:2927-31;and Yeh et al. (1982) Int. J. Cancer 29:269-75), the more recent human Bcell hybridoma technique (Kozbor et al. (1983) Immunol. Today 4:72), theEBV-hybridoma technique (Cole et al. (1985), Monoclonal Antibodies andCancer Therapy, Alan R. Liss, Inc., pp. 77-96) or trioma techniques. Thetechnology for producing monoclonal antibody hybridomas is well known(see generally R. H. Kenneth, in Monoclonal Antibodies: A New DimensionIn Biological Analyses, Plenum Publishing Corp., New York, N.Y. (1980);E. A. Lerner (1981) Yale J. Biol. Med., 54:387-402; M. L. Gefter et al.(1977) Somatic Cell Genet. 3:231-36). Briefly, an immortal cell line(typically a myeloma) is fused to lymphocytes (typically splenocytes)from a mammal immunized with a BAL1 and/or BBAP immunogen as describedabove, and the culture supernatants of the resulting hybridoma cells arescreened to identify a hybridoma producing a monoclonal antibody thatbinds BAL1 and/or BBAP.

Any of the many well-known protocols used for fusing lymphocytes andimmortalized cell lines can be applied for the purpose of generating ananti-BAL1 and/or BBAP monoclonal antibody (see, i.e., G. Galfre et al.(1977) Nature 266:550-52; Gefter et al. Somatic Cell Genet., citedsupra; Lerner, Yale J. Biol. Med., cited supra; Kenneth, MonoclonalAntibodies, cited supra). Moreover, the ordinarily skilled worker willappreciate that there are many variations of such methods which alsowould be useful. Typically, the immortal cell line (e.g., a myeloma cellline) is derived from the same mammalian species as the lymphocytes. Forexample, murine hybridomas can be made by fusing lymphocytes from amouse immunized with an immunogenic preparation of the present inventionwith an immortalized mouse cell line. Preferred immortal cell lines aremouse myeloma cell lines that are sensitive to culture medium containinghypoxanthine, aminopterin and thymidine (“HAT medium”). Any of a numberof myeloma cell lines can be used as a fusion partner according tostandard techniques, i.e., the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 orSp2/O-Ag14 myeloma lines. These myeloma lines are available from ATCC.Typically, HAT-sensitive mouse myeloma cells are fused to mousesplenocytes using polyethylene glycol (“PEG”). Hybridoma cells resultingfrom the fusion are then selected using HAT medium, which kills unfusedand unproductively fused myeloma cells (unfused splenocytes die afterseveral days because they are not transformed). Hybridoma cellsproducing a monoclonal antibody of the invention are detected byscreening the hybridoma culture supernatants for antibodies that bindBAL1 and/or BBAP, i.e., using a standard ELISA assay.

As an alternative to preparing monoclonal antibody-secreting hybridomas,a monoclonal anti-BAL1 and/or BBAP antibody can be identified andisolated by screening a recombinant combinatorial immunoglobulin library(e.g., an antibody phage display library) with BAL1 and/or BBAP tothereby isolate immunoglobulin library members that bind BAL1 and/orBBAP. Kits for generating and screening phage display libraries arecommercially available (e.g., the Pharmacia Recombinant Phage AntibodySystem, Catalog No. 27-9400-01; and the Stratagene SurfZAP™ PhageDisplay Kit, Catalog No. 240612). Additionally, examples of methods andreagents particularly amenable for use in generating and screeningantibody display library can be found in, for example, Ladner et al.U.S. Pat. No. 5,223,409; Kang et al. PCT International Publication No.WO 92/18619; Dower et al. PCT International Publication No. WO 91/17271;Winter et al. PCT International Publication WO 92/20791; Markland et al.PCT International Publication No. WO 92/15679; Breitling et al. PCTInternational Publication WO 93/01288; McCafferty et al. PCTInternational Publication No. WO 92/01047; Garrard et al. PCTInternational Publication No. WO 92/09690; Ladner et al. PCTInternational Publication No. WO 90/02809; Fuchs et al. (1991)Bio/Technology 9:1369-1372; Hay et al. (1992) Hum. Antibod. Hybridomas3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffiths et al.(1993) EMBO J. 12:725-734; Hawkins et al. (1992) J. Mol. Biol.226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al.(1992) Proc. Natl. Acad. Sci. USA 89:3576-3580; Garrard et al. (1991)Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nucleic Acids Res.19:4133-4137; Barbas et al. (1991) Proc. Natl. Acad. Sci. USA88:7978-7982; and McCafferty et al. Nature (1990) 348:552-554.

Additionally, recombinant anti-BAL1 and/or BBAP antibodies, such aschimeric and humanized monoclonal antibodies, comprising both human andnon-human portions, which can be made using standard recombinant DNAtechniques, are within the scope of the invention. Such chimeric andhumanized monoclonal antibodies can be produced by recombinant DNAtechniques known in the art, for example using methods described inRobinson et al. International Application No. PCT/US86/02269; Akira, etal. European Patent Application 184,187; Taniguchi, M., European PatentApplication 171,496; Morrison et al. European Patent Application173,494; Neuberger et al. PCT International Publication No. WO 86/01533;Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly et al. European PatentApplication 125,023; Better et al. (1988) Science 240:1041-1043; Liu etal. (1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J.Immunol. 139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA84:214-218; Nishimura et al. (1987) Canc. Res. 47:999-1005; Wood et al.(1985) Nature 314:446-449; and Shaw et al. (1988) J. Natl. Cancer Inst.80:1553-1559); Morrison, S. L. (1985) Science 229:1202-1207; Oi et al.(1986) BioTechniques 4:214; Winter U.S. Pat. No. 5,225,539; Jones et al.(1986) Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; andBeidler et al. (1988) J. Immunol. 141:4053-4060.

An anti-BAL1 and/or BBAP antibody (e.g., monoclonal antibody) can beused to isolate BAL1 and/or BBAP by standard techniques, such asaffinity chromatography or immunoprecipitation. An anti-BAL1 and/or BBAPantibody can facilitate the purification of natural BAL1 and/or BBAPfrom cells and of recombinantly produced BAL1 and/or BBAP expressed inhost cells. Moreover, an anti-BAL1 and/or BBAP antibody can be used todetect BAL1 and/or BBAP protein (e.g., in a cellular lysate or cellsupernatant) in order to evaluate the abundance and pattern ofexpression of the BAL1 and/or BBAP protein. In some embodiments, forexample, such antibodies can be used in quantitative immunohistochemicalassays to determine or predict the efficacy of a cancer therapy. Thus,anti-BAL1 and/or BBAP antibodies can be used to monitor protein levelsin a cell or tissue, e.g., cancer cells or tissue, as part of a clinicaltesting procedure, e.g., in order to monitor the efficacy of a cancertherapy. Detection can be facilitated by coupling (e.g., physicallylinking) the antibody to a detectable substance. Examples of detectablesubstances include various enzymes, prosthetic groups, fluorescentmaterials, luminescent materials, bioluminescent materials, andradioactive materials. Examples of suitable enzymes include horseradishperoxidase, alkaline phosphatase, β-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin, and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ³⁵S or ³H.

In vivo techniques for detection of BAL1 and/or BBAP protein includeintroducing into a subject a labeled antibody directed against theprotein. For example, the antibody can be labeled with a radioactivemarker whose presence and location in a subject can be detected bystandard imaging techniques.

IV. Identification of Compounds that Modulate BAL1 and/or BBAP

The BAL1 and/or BBAP nucleic acid and polypeptide molecules describedherein may be used to design modulators of one or more of biologicalactivities of the complex or complex polypeptides. In particular,information useful for the design of therapeutic and diagnosticmolecules, including, for example, the protein domain, structuralinformation, and the like for polypeptides of the invention is nowavailable or attainable as a result of the ability to prepare, purifyand characterize the complexes and complex polypeptides, and domains,fragments, variants and derivatives thereof.

In one aspect, modulators, inhibitors, or antagonists against thepolypeptides of the invention, biological complexes containing them, ororthologues thereof, may be used to treat any disease or other treatablecondition of a patient (including humans and animals), including, forexample, cancer.

Modulators of BAL1 and/or BBAP nucleic acid and polypeptide molecules,may be identified and developed as set forth below using techniques andmethods known to those of skill in the art. The modulators of theinvention may be employed, for instance, to inhibit and treat BAL1and/or BBAP-mediated diseases or disorders. The modulators of theinvention may elicit a change in one or more of the followingactivities: (a) a change in the level and/or rate of formation of aBAL1-BBAP complex, (b) a change in the activity of a BAL1 and/or BBAPnucleic acid and/or polypeptide, (c) a change in the stability of a BAL1and/or BBAP nucleic acid and/or polypeptide, (d) a change in theconformation of a BAL1 and/or BBAP nucleic acid and/or polypeptide, or(e) a change in the activity of at least one polypeptide contained in aBAL1-BBAP complex. A number of methods for identifying a molecule whichmodulates a BAL1 and/or BBAP nucleic acid and/or polypeptide are knownin the art. For example, in one such method, a BAL1 and/or BBAP nucleicacid and/or polypeptide, is contacted with a test compound, and theactivity of the BAL1 and/or BBAP nucleic acid and/or polypeptide isdetermined in the presence of the test compound, wherein a change in theactivity of the BAL1 and/or BBAP nucleic acid and/or polypeptide in thepresence of the compound as compared to the activity in the absence ofthe compound (or in the presence of a control compound) indicates thatthe test compound modulates the activity of the BAL1 and/or BBAP nucleicacid and/or polypeptide.

Compounds to be tested for their ability to act as modulators of BAL1and/or BBAP nucleic acids and/or polypeptides, can be produced, forexample, by bacteria, yeast or other organisms (e.g. natural products),produced chemically (e.g. small molecules, including peptidomimetics),or produced recombinantly. Compounds for use with the above-describedmethods may be selected from the group of compounds consisting oflipids, carbohydrates, polypeptides, peptidomimetics, peptide-nucleicacids (PNAs), small molecules, natural products, aptamers andpolynucleotides. In certain embodiments, the compound is apolynucleotide. In some embodiments, said polynucleotide is an antisensenucleic acid. In other embodiments, said polynucleotide is an siRNA. Incertain embodiments, the compound comprises a biologically activefragment of a BAL1 and/or BBAP polypeptide (e.g., a dominant negativeform that binds to, but does not activate, BAL1 and/or BBAP).

A variety of assay formats will suffice and, in light of the presentdisclosure, those not expressly described herein may nevertheless becomprehended by one of ordinary skill in the art based on the teachingsherein. Assay formats for analyzing BAL1-BBAP complex formation and/oractivity of a BAL1 and/or BBAP nucleic acid and/or polypeptide, may begenerated in many different forms, and include assays based on cell-freesystems, e.g. purified proteins or cell lysates, as well as cell-basedassays which utilize intact cells. Simple binding assays can also beused to detect agents which modulate a BAL1 and/or BBAP, for example, byenhancing the formation of a BAL1 and/or BBAP, by enhancing the bindingof a BAL1 and/or BBAP to a substrate, and/or by enhancing the binding ofa BAL1 and/or BBAP polypeptide to a substrate. Another example of anassay useful for identifying a modulator of a BAL1 and/or BBAP is acompetitive assay that combines one or more BAL1 and/or BBAPpolypeptides with a potential modulator, such as, for example,polypeptides, nucleic acids, natural substrates or ligands, or substrateor ligand mimetics, under appropriate conditions for a competitiveinhibition assay. BAL1 and/or BBAP polypeptides can be labeled, such asby radioactivity or a colorimetric compound, such that BAL1 and/or BBAPcomplex formation and/or activity can be determined accurately to assessthe effectiveness of the potential modulator.

Assays may employ kinetic or thermodynamic methodology using a widevariety of techniques including, but not limited to, microcalorimetry,circular dichroism, capillary zone electrophoresis, nuclear magneticresonance spectroscopy, fluorescence spectroscopy, and combinationsthereof. Assays may also employ any of the methods for isolating,preparing and detecting BAL1 and/or BBAPes polypeptides or complexes, asdescribed above.

Complex formation between a BAL1 and/or BBAP polypeptide, or fragmentthereof, and a binding partner (e.g., BAL1 and/or BBAP) may be detectedby a variety of methods. Modulation of the complex's formation may bequantified using, for example, detectably labeled proteins such asradiolabeled, fluorescently labeled, or enzymatically labeledpolypeptides or binding partners, by immunoassay, or by chromatographicdetection. Methods of isolating and identifying BAL1-BBAP complexesdescribed above may be incorporated into the detection methods.

In certain embodiments, it may be desirable to immobilize a BAL1 and/orBBAP polypeptide to facilitate separation of BAL1-BBAP complexes fromuncomplexed forms of one or both of the proteins, as well as toaccommodate automation of the assay. Binding of a BAL1 and/or BBAPpolypeptide to a binding partner may be accomplished in any vesselsuitable for containing the reactants. Examples include microtitreplates, test tubes, and micro-centrifuge tubes. In one embodiment, afusion protein may be provided which adds a domain that allows theprotein to be bound to a matrix. For example,glutathione-S-transferase/polypeptide (GST/polypeptide) fusion proteinsmay be adsorbed onto glutathione sepharose beads (Sigma Chemical, St.Louis, Mo.) or glutathione derivatized microtitre plates, which are thencombined with the binding partner, e.g. an ³⁵S-labeled binding partner,and the test compound, and the mixture incubated under conditionsconducive to complex formation, e.g. at physiological conditions forsalt and pH, though slightly more stringent conditions may be desired.Following incubation, the beads are washed to remove any unbound label,and the matrix immobilized and radiolabel determined directly (e.g.beads placed in scintillant), or in the supernatant after the complexesare subsequently dissociated. Alternatively, the complexes may bedissociated from the matrix, separated by SDS-PAGE, and the level ofBAL1 and/or BBAP polypeptides found in the bead fraction quantified fromthe gel using standard electrophoretic techniques such as described inthe appended examples.

Other techniques for immobilizing proteins on matrices are alsoavailable for use in the subject assay. For instance, a BAL1 and/or BBAPpolypeptide may be immobilized utilizing conjugation of biotin andstreptavidin. For instance, biotinylated polypeptide molecules may beprepared from biotin-NHS(N-hydroxy-succinimide) using techniques wellknown in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford,Ill.), and immobilized in the wells of streptavidin-coated 96 wellplates (Pierce Chemical). Alternatively, antibodies reactive with thepolypeptide may be derivatized to the wells of the plate, andpolypeptide trapped in the wells by antibody conjugation. As above,preparations of a binding partner and a test compound are incubated inthe polypeptide presenting wells of the plate, and the amount of complextrapped in the well may be quantified. Exemplary methods for detectingsuch complexes, in addition to those described above for theGST-immobilized complexes, include immunodetection of complexes usingantibodies reactive with the binding partner, or which are reactive withthe BAL1 and/or BBAP polypeptide and compete with the binding partner;as well as enzyme-linked assays which rely on detecting an enzymaticactivity associated with the binding partner, either intrinsic orextrinsic activity. In the instance of the latter, the enzyme may bechemically conjugated or provided as a fusion protein with the bindingpartner. To illustrate, the binding partner may be chemicallycross-linked or genetically fused with horseradish peroxidase, and theamount of BAL1 and/or BBAP polypeptide trapped in the BAL1-BBAP complexmay be assessed with a chromogenic substrate of the enzyme, e.g.3,3′-diamino-benzadine tetrahydrochloride or 4-chloro-1-naphthol.Likewise, a fusion protein comprising the BAL1 and/or BBAP polypeptideand glutathione-S-transferase may be provided, and BAL1-BBAP complexformation quantified by detecting the GST activity using1-chloro-2,4-dinitrobenzene (Habig et al (1974) J Biol Chem 249:7130).

Antibodies against the BAL1 and/or BBAP polypeptide can be used forimmunodetection purposes. Alternatively, the BAL1 and/or BBAPpolypeptide to be detected may be “epitope-tagged” in the form of afusion protein that includes, in addition to the polypeptide sequence, asecond polypeptide for which antibodies are readily available (e.g. fromcommercial sources). For instance, the GST fusion proteins describedabove may also be used for quantification of binding using antibodiesagainst the GST moiety. Other useful epitope tags include myc-epitopes(e.g., see Ellison et al. (1991) J Biol Chem 266:21150-21157) whichincludes a 10-residue sequence from c-myc, as well as the pFLAG system(International Biotechnologies, Inc.) or the pEZZ-protein A system(Pharmacia, N.J.).

In certain in vitro embodiments of the present assay, the protein or theset of proteins engaged in a protein-protein, protein-substrate, orprotein-nucleic acid interaction comprises a reconstituted proteinmixture of at least semi-purified proteins. By semi-purified, it ismeant that the proteins utilized in the reconstituted mixture have beenpreviously separated from other cellular or viral proteins. Forinstance, in contrast to cell lysates, the proteins involved in aprotein-substrate, protein-protein or nucleic acid-protein interactionare present in the mixture to at least 50% purity relative to all otherproteins in the mixture, and more preferably are present at 90-95%purity. In certain embodiments of the subject method, the reconstitutedprotein mixture is derived by mixing highly purified proteins such thatthe reconstituted mixture substantially lacks other proteins (such as ofcellular or viral origin) which might interfere with or otherwise alterthe ability to measure activity resulting from the givenprotein-substrate, protein-protein interaction, or nucleic acid-proteininteraction.

In one embodiment, the use of reconstituted protein mixtures allows morecareful control of the protein-substrate, protein-protein, or nucleicacid-protein interaction conditions. Moreover, the system may be derivedto favor discovery of modulators of particular intermediate states ofthe protein-protein interaction. For instance, a reconstituted proteinassay may be carried out both in the presence and absence of a candidateagent, thereby allowing detection of a modulator of a givenprotein-substrate, protein-protein, or nucleic acid-protein interaction.

Assaying biological activity resulting from a given protein-substrate,protein-protein or nucleic acid-protein interaction, in the presence andabsence of a candidate modulator, may be accomplished in any vesselsuitable for containing the reactants. Examples include microtitreplates, test tubes, and micro-centrifuge tubes.

In yet another embodiment, a BAL1 and/or BBAP polypeptide may be used togenerate a two-hybrid or interaction trap assay (see also, U.S. Pat. No.5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J.Biol Chem 268:12046-12054; Bartel et al. (1993) Biotechniques14:920-924; and Iwabuchi et al. (1993) Oncogene 8:1693-1696), forsubsequently detecting agents which disrupt binding of the interactioncomponents to one another.

In particular, the method makes use of chimeric genes which expresshybrid proteins. To illustrate, a first hybrid gene comprises the codingsequence for a DNA binding domain of a transcriptional activator may befused in frame to the coding sequence for a “bait” protein, e.g., a BAL1and/or BBAP polypeptide of sufficient length to bind to a potentialinteracting protein. The second hybrid protein encodes a transcriptionalactivation domain fused in frame to a gene encoding a “fish” protein,e.g., a potential interacting protein of sufficient length to interactwith the protein-protein interaction component polypeptide portion ofthe bait fusion protein. If the bait and fish proteins are able tointeract, e.g., form a protein-protein interaction component complex,they bring into close proximity the two domains of the transcriptionalactivator. This proximity causes transcription of a reporter gene whichis operably linked to a transcriptional regulatory site responsive tothe transcriptional activator, and expression of the reporter gene maybe detected and used to score for the interaction of the bait and fishproteins. The host cell also contains a first chimeric gene which iscapable of being expressed in the host cell. The gene encodes a chimericprotein, which comprises (a) a DNA binding domain that recognizes theresponsive element on the reporter gene in the host cell, and (b) a baitprotein (e.g., a BAL1 and/or BBAP polypeptide). A second chimeric geneis also provided which is capable of being expressed in the host cell,and encodes the “fish” fusion protein. In one embodiment, both the firstand the second chimeric genes are introduced into the host cell in theform of plasmids. Preferably, however, the first chimeric gene ispresent in a chromosome of the host cell and the second chimeric gene isintroduced into the host cell as part of a plasmid.

The DNA binding domain of the first hybrid protein and thetranscriptional activation domain of the second hybrid protein may bederived from transcriptional activators having separable DNA binding andtranscriptional activation domains. For instance, these separate DNAbinding and transcriptional activation domains are known to be found inthe yeast GAL4 protein, and are known to be found in the yeast GCN4 andADR1 proteins. Many other proteins involved in transcription also haveseparable binding and transcriptional activation domains which make themuseful for the present invention, and include, for example, the LexA andVP16 proteins. It will be understood that other (substantially)transcriptionally-inert DNA binding domains may be used in the subjectconstructs; such as domains of ACE1, λcI, lac repressor, jun or fos. Inanother embodiment, the DNA binding domain and the transcriptionalactivation domain may be from different proteins. The use of a LexA DNAbinding domain provides certain advantages. For example, in yeast, theLexA moiety contains no activation function and has no known affect ontranscription of yeast genes. In addition, use of LexA allows controlover the sensitivity of the assay to the level of interaction (see, forexample, the Brent et al. PCT publication WO94/10300).

In certain embodiments, any enzymatic activity associated with the baitor fish proteins is inactivated, e.g., dominant negative or othermutants of a protein-protein interaction component can be used.

Continuing with the illustrative example, formation of a complex betweenthe bait and fish fusion proteins in the host cell, causes theactivation domain to activate transcription of the reporter gene. Themethod is carried out by introducing the first chimeric gene and thesecond chimeric gene into the host cell, and subjecting that cell toconditions under which the bait and fish fusion proteins and areexpressed in sufficient quantity for the reporter gene to be activated.The formation of a complex results in a detectable signal produced bythe expression of the reporter gene.

In still further embodiments, the BAL1 and/or BBAP, or complexpolypeptide, of interest may be generated in whole cells, takingadvantage of cell culture techniques to support the subject assay. Forexample, the BAL1 and/or BBAP, or complex polypeptide, may beconstituted in a prokaryotic or eukaryotic cell culture system.Advantages to generating the BAL1 and/or BBAP, or complex polypeptide,in an intact cell includes the ability to screen for modulators of thelevel and/or activity of the BAL1 and/or BBAP, or complex polypeptide,which are functional in an environment more closely approximating thatwhich therapeutic use of the modulator would require, including theability of the agent to gain entry into the cell. Furthermore, certainof the in vivo embodiments of the assay are amenable to high through-putanalysis of candidate agents.

The BAL1 and/or BBAP nucleic acids and/or polypeptide can be endogenousto the cell selected to support the assay. Alternatively, some or all ofthe components can be derived from exogenous sources. For instance,fusion proteins can be introduced into the cell by recombinanttechniques (such as through the use of an expression vector), as well asby microinjecting the fusion protein itself or mRNA encoding the fusionprotein. Moreover, in the whole cell embodiments of the subject assay,the reporter gene construct can provide, upon expression, a selectablemarker. Such embodiments of the subject assay are particularly amenableto high through-put analysis in that proliferation of the cell canprovide a simple measure of the protein-protein interaction.

The amount of transcription from the reporter gene may be measured usingany method known to those of skill in the art to be suitable. Forexample, specific mRNA expression may be detected using Northern blotsor specific protein product may be identified by a characteristic stain,western blots or an intrinsic activity. In certain embodiments, theproduct of the reporter gene is detected by an intrinsic activityassociated with that product. For instance, the reporter gene may encodea gene product that, by enzymatic activity, gives rise to a detectionsignal based on color, fluorescence, or luminescence.

In many drug screening programs which test libraries of compounds andnatural extracts, high throughput assays are desirable in order tomaximize the number of compounds surveyed in a given period of time.Assays of the present invention which are performed in cell-freesystems, such as may be derived with purified or semi-purified proteinsor with lysates, are often preferred as “primary” screens in that theycan be generated to permit rapid development and relatively easydetection of an alteration in a molecular target which is mediated by atest compound. Moreover, the effects of cellular toxicity and/orbioavailability of the test compound can be generally ignored in the invitro system, the assay instead being focused primarily on the effect ofthe drug on the molecular target as may be manifest in an alteration ofbinding affinity with other proteins or changes in enzymatic propertiesof the molecular target. Accordingly, potential modulators of BAL1and/or BBAP may be detected in a cell-free assay generated byconstitution of a functional BAL1 and/or BBAP in a cell lysate. In analternate format, the assay can be derived as a reconstituted proteinmixture which, as described below, offers a number of benefits overlysate-based assays.

The activity of a BAL1 and/or BBAP or a BAL1 and/or BBAP polypeptide maybe identified and/or assayed using a variety of methods well known tothe skilled artisan. For example, the activity of a BAL1 and/or BBAPnucleic acid and/or polypeptide may be determined by assaying for thelevel of expression of RNA and/or protein molecules. Transcriptionlevels may be determined, for example, using Northern blots,hybridization to an oligonucleotide array or by assaying for the levelof a resulting protein product. Translation levels may be determined,for example, using Western blotting or by identifying a detectablesignal produced by a protein product (e.g., fluorescence, luminescence,enzymatic activity, etc.). Depending on the particular situation, it maybe desirable to detect the level of transcription and/or translation ofa single gene or of multiple genes.

In other embodiments, the biological activity of a BAL1 and/or BBAPnucleic acid and/or polypeptide may be assessed by monitoring changes inthe phenotype of a targeted cell. For example, the detection means caninclude a reporter gene construct which includes a transcriptionalregulatory element that is dependent in some form on the level and/oractivity of a BAL1 and/or BBAP nucleic acid and/or polypeptide. The BAL1and/or BBAP nucleic acid and/or polypeptide may be provided as a fusionprotein with a domain that binds to a DNA element of a reporter geneconstruct. The added domain of the fusion protein can be one which,through its DNA binding ability, increases or decreases transcription ofthe reporter gene. Whichever the case may be, its presence in the fusionprotein renders it responsive to a BAL1 and/or BBAP nucleic acid and/orpolypeptide. Accordingly, the level of expression of the reporter genewill vary with the level of expression of a BAL1 and/or BBAP nucleicacid and/or polypeptide.

Moreover, in the whole cell embodiments of the subject assay, thereporter gene construct can provide, upon expression, a selectablemarker. A reporter gene includes any gene that expresses a detectablegene product, which may be RNA or protein. Preferred reporter genes arethose that are readily detectable. The reporter gene may also beincluded in the construct in the form of a fusion gene with a gene thatincludes desired transcriptional regulatory sequences or exhibits otherdesirable properties. For instance, the product of the reporter gene canbe an enzyme which confers resistance to an antibiotic or other drug, oran enzyme which complements a deficiency in the host cell (i.e.thymidine kinase or dihydrofolate reductase). To illustrate, theaminoglycoside phosphotransferase encoded by the bacterial transposongene Tn5 neo can be placed under transcriptional control of a promoterelement responsive to the level of a BAL1 and/or BBAP nucleic acidand/or polypeptide present in the cell. Such embodiments of the subjectassay are particularly amenable to high through-put analysis in thatproliferation of the cell can provide a simple measure of inhibition ofthe BAL1 and/or BBAP nucleic acid and/or polypeptide.

V. Methods of the Invention

The methods of the invention relate to the modulation of the expressionand/or activity of BAL1 and/or BBAP sufficient to inhibit proliferationof hyperproliferative cells and to treat cancer. In some embodiments,this can occur in vivo (e.g., within a subject). In other embodiments,this can occur in vitro (e.g., within a medium such as a cell culturemedium, body fluid sample, tissue sample, etc. containinghyperproliferative cells). In still other embodiments, medicamentscomprising agents useful for such inhibition are contemplated.

The term “sufficient to inhibit” is intended to encompass any decreasein expression and/or activity of BAL1 and/or BBAP that promotes,activates, stimulates, enhances, or results in inhibition ofproliferation of hyperproliferative cells and/or treatment of cancer.

In one aspect, a method of treating cancer by enhancing the efficacy ofcancer therapies in a subject, comprising administering to the subjectan effective amount of (a) an agent that inhibits one or more functionsof BAL1, BBAP, or a BAL1-BBAP complex and (b) the cancer therapy isprovided. In one embodiment, the agent is a recombinant BAL1 and/or BBAPprotein, or fragment thereof, or nucleic acid molecule encoding such apolypeptide. In another embodiment, the agent is an anti-sense nucleicacid molecule having a sequence complementary to BAL1 and/or BBAP (e.g.,an RNAi, siRNA, or other RNA inhibiting nucleic acid molecule). In stillother embodiments, the agent is a small molecule which inhibits activityof the marker; an aptamer which inhibits expression or activity of themarker. In yet other embodiments, the agent is a polypeptide describedherein (e.g., a dominant negative BAL1 and/or BBAP polypeptide).

The term “administering” is intended to include routes of administrationwhich allow the agent to perform its intended function of modulating(e.g., increasing or decreasing) expression and/or activity of BAL1and/or BBAP. Examples of routes of administration which can be usedinclude injection (subcutaneous, intravenous, parenterally,intraperitoneally, intrathecal, etc., such as in a subcutaneousinjection into white fate depots), oral, inhalation, and transdermal.The injection can be bolus injections or can be continuous infusion.Depending on the route of administration, the agent can be coated withor disposed in a selected material to protect it from natural conditionswhich may detrimentally affect its ability to perform its intendedfunction. The agent may be administered alone, or in conjunction with apharmaceutically acceptable carrier. Further the agent may becoadministered with a pharmaceutically acceptable carrier. The agentalso may be administered as a prodrug, which is converted to its activeform in vivo. The agent may also be administered in combination with oneor more additional therapeutic agent(s) (e.g., before, after orsimultaneously therewith).

The term “effective amount” of an agent that induces expression and/oractivity of BAL1 and/or BBAP is that amount necessary or sufficient tomodulate (e.g., increase or decrease) expression and/or activity of BAL1and/or BBAP in the subject or population of subjects. The effectiveamount can vary depending on such factors as the type of therapeuticagent(s) employed, the size of the subject, or the severity of thedisorder.

It will be appreciated that individual dosages may be varied dependingupon the requirements of the subject in the judgment of the attendingclinician, the severity of the condition being treated and theparticular compound being employed. In determining the therapeuticallyeffective amount or dose, a number of additional factors may beconsidered by the attending clinician, including, but not limited to:the pharmacodynamic characteristics of the particular respirationuncoupling agent and its mode and route of administration; the desiredtime course of treatment; the species of mammal; its size, age, andgeneral health; the specific disease involved; the degree of orinvolvement or the severity of the disease; the response of theindividual subject; the particular compound administered; the mode ofadministration; the bioavailability characteristics of the preparationadministered; the dose regimen selected; the kind of concurrenttreatment; and other relevant circumstances.

Treatment can be initiated with smaller dosages which are less than theeffective dose of the compound. Thereafter, in one embodiment, thedosage should be increased by small increments until the optimum effectunder the circumstances is reached. For convenience, the total dailydosage may be divided and administered in portions during the day ifdesired.

The effectiveness of any particular respiration agent to treat a cancercan be monitored by comparing two or more samples obtained from asubject undergoing cancer therapy. For example, the efficacy of cancertherapies which damage DNA, as well as agents that take advantage of DNArepair defects but do not damage DNA themselves, such as poly ADP ribosepolymerase (PARP) inhibitors, as well as chemotherapy or radiationtherapy, is predicted according to the methods described herein.

In one embodiment, the efficacy of chemotherapies is predicted.Chemotherapy includes the administration of a chemotherapeutic agent.Such a chemotherapeutic agent may be, but is not limited to, thoseselected from among the following groups of compounds: platinumcompounds, cytotoxic antibiotics, antimetabolites, anti-mitotic agents,alkylating agents, arsenic compounds, DNA topoisomerase inhibitors,taxanes, nucleoside analogues, plant alkaloids, and toxins; andsynthetic derivatives thereof. Exemplary compounds include, but are notlimited to, alkylating agents: cisplatin, treosulfan, and trofosfamide;plant alkaloids: vinblastine, paclitaxel, docetaxol; DNA topoisomeraseinhibitors: teniposide, crisnatol, and mitomycin; anti-folates:methotrexate, mycophenolic acid, and hydroxyurea; pyrimidine analogs:5-fluorouracil, doxifluridine, and cytosine arabinoside; purine analogs:mercaptopurine and thioguanine; DNA antimetabolites:2′-deoxy-5-fluorouridine, aphidicolin glycinate, and pyrazoloimidazole;and antimitotic agents: halichondrin, colchicine, and rhizoxin.Compositions comprising one or more chemotherapeutic agents (e.g., FLAG,CHOP) may also be used. FLAG comprises fludarabine, cytosine arabinoside(Ara-C) and G-CSF. CHOP comprises cyclophosphamide, vincristine,doxorubicin, and prednisone. In another embodiments, PARP (e.g., PARP-1and/or PARP-2) inhibitors are used and such inhibitors are well known inthe art (e.g., Olaparib, ABT-888, BSI-201, BGP-15 (N-Gene ResearchLaboratories, Inc.); INO-1001 (Inotek Pharmaceuticals Inc.); PJ34(Soriano et al., 2001; Pacher et al., 2002b); 3-aminobenzamide(Trevigen); 4-amino-1,8-naphthalimide; (Trevigen);6(5H)-phenanthridinone (Trevigen); benzamide (U.S. Pat. Re. 36,397); andNU1025 (Bowman et al.). The foregoing examples of chemotherapeuticagents are illustrative, and are not intended to be limiting.

In another embodiment, the methods described herein are useful forpredicting the efficacy of radiation therapy. The radiation used inradiation therapy can be ionizing radiation. Radiation therapy can alsobe gamma rays, X-rays, or proton beams. Examples of radiation therapyinclude, but are not limited to, external-beam radiation therapy,interstitial implantation of radioisotopes (1-125, palladium, iridium),radioisotopes such as strontium-89, thoracic radiation therapy,intraperitoneal P-32 radiation therapy, and/or total abdominal andpelvic radiation therapy. For a general overview of radiation therapy,see Hellman, Chapter 16: Principles of Cancer Management: RadiationTherapy, 6th edition, 2001, DeVita et al., eds., J. B. LippencottCompany, Philadelphia. The radiation therapy can be administered asexternal beam radiation or teletherapy wherein the radiation is directedfrom a remote source. The radiation treatment can also be administeredas internal therapy or brachytherapy wherein a radioactive source isplaced inside the body close to cancer cells or a tumor mass. Alsoencompassed is the use of photodynamic therapy comprising theadministration of photosensitizers, such as hematoporphyrin and itsderivatives, Vertoporfin (BPD-MA), phthalocyanine, photosensitizer Pc4,demethoxy-hypocrellin A; and 2BA-2-DMHA.

Cancer therapies which damage DNA to a lesser extent than chemotherapyor radiation therapy may be determined to have enhanced efficacy usingthe methods of the invention for determining the phenotype of a cancer.Examples of such therapies include immunotherapy, hormone therapy, andgene therapy. In addition, other such therapies are known in the art,including hyperthermic therapy and photodynamic therapy (see NationalCancer Institute home page at nci.nih.gov). Such therapies include, butare not limited to, the use of antisense polynucleotides, ribozymes, RNAinterference molecules, triple helix polynucleotides and the like, wherethe nucleotide sequence of such compounds are related to the nucleotidesequences of DNA and/or RNA of genes that are linked to the initiation,progression, and/or pathology of a tumor or cancer. For example,oncogenes, growth factor genes, growth factor receptor genes, cell cyclegenes, DNA repair genes, and others, may be used in such therapies.

Immunotherapy may comprise, for example, use of cancer vaccines and/orsensitized antigen presenting cells. The immunotherapy can involvepassive immunity for short-term protection of a host, achieved by theadministration of pre-formed antibody directed against a cancer antigenor disease antigen (e.g., administration of a monoclonal antibody,optionally linked to a chemotherapeutic agent or toxin, to a tumorantigen). Immunotherapy can also focus on using the cytotoxiclymphocyte-recognized epitopes of cancer cell lines.

Hormonal therapeutic treatments can comprise, for example, hormonalagonists, hormonal antagonists (e.g., flutamide, bicalutamide,tamoxifen, raloxifene, leuprolide acetate (LUPRON), LH-RH antagonists),inhibitors of hormone biosynthesis and processing, and steroids (e.g.,dexamethasone, retinoids, deltoids, betamethasone, cortisol, cortisone,prednisone, dehydrotestosterone, glucocorticoids, mineralocorticoids,estrogen, testosterone, progestins), vitamin A derivatives (e.g.,all-trans retinoic acid (ATRA)); vitamin D3 analogs; antigestagens(e.g., mifepristone, onapristone), or antiandrogens (e.g., cyproteroneacetate).

In one embodiment, cancer therapy used for cancers whose phenotype isdetermined by the methods of the invention can comprise one or moretypes of therapies described herein including, but not limited to,chemotherapeutic agents, immunotherapeutics, anti-angiogenic agents,cytokines, hormones, antibodies, polynucleotides, radiation andphotodynamic therapeutic agents. For example, combination therapies cancomprise one or more chemotherapeutic agents and radiation, one or morechemotherapeutic agents and immunotherapy, or one or morechemotherapeutic agents, radiation and chemotherapy.

Hyperthermia, a procedure in which body tissue is exposed to hightemperatures (up to 106° F.), is under investigation to assess itseffectiveness in the treatment of cancer. Heat may help shrink tumors bydamaging cells or depriving them of substances they need to live.Hyperthermia therapy can be local, regional, and whole-bodyhyperthermia, using external and internal heating devices. Hyperthermiais almost always used with other forms of therapy (e.g., radiationtherapy, chemotherapy, and biological therapy) to try to increase theireffectiveness.

Local hyperthermia refers to heat that is applied to a very small area,such as a tumor. The area may be heated externally with high-frequencywaves aimed at a tumor from a device outside the body. To achieveinternal heating, one of several types of sterile probes may be used,including thin, heated wires or hollow tubes filled with warm water;implanted microwave antennae; and radiofrequency electrodes. In regionalhyperthermia, an organ or a limb is heated. Magnets and devices thatproduce high energy are placed over the region to be heated. In anotherapproach, called perfusion, some of the patient's blood is removed,heated, and then pumped (perfused) into the region that is to be heatedinternally. Whole-body heating is used to treat metastatic cancer thathas spread throughout the body. It can be accomplished using warm-waterblankets, hot wax, inductive coils (like those in electric blankets), orthermal chambers (similar to large incubators). Hyperthermia does notcause any marked increase in radiation side effects or complications.Heat applied directly to the skin, however, can cause discomfort or evensignificant local pain in about half the patients treated. It can alsocause blisters, which generally heal rapidly.

Photodynamic therapy (also called PDT, photoradiation therapy,phototherapy, or photochemotherapy) is a treatment for some types ofcancer. It is based on the discovery that certain chemicals known asphotosensitizing agents can kill one-celled organisms when the organismsare exposed to a particular type of light. PDT destroys cancer cellsthrough the use of a fixed-frequency laser light in combination with aphotosensitizing agent. In PDT, the photosensitizing agent is injectedinto the bloodstream and absorbed by cells all over the body. The agentremains in cancer cells for a longer time than it does in normal cells.When the treated cancer cells are exposed to laser light, thephotosensitizing agent absorbs the light and produces an active form ofoxygen that destroys the treated cancer cells. Light exposure must betimed carefully so that it occurs when most of the photosensitizingagent has left healthy cells but is still present in the cancer cells.The laser light used in PDT can be directed through a fiber-optic (avery thin glass strand). The fiber-optic is placed close to the cancerto deliver the proper amount of light. The fiber-optic can be directedthrough a bronchoscope into the lungs for the treatment of lung canceror through an endoscope into the esophagus for the treatment ofesophageal cancer. An advantage of PDT is that it causes minimal damageto healthy tissue. However, because the laser light currently in usecannot pass through more than about 3 centimeters of tissue (a littlemore than one and an eighth inch), PDT is mainly used to treat tumors onor just under the skin or on the lining of internal organs. Photodynamictherapy makes the skin and eyes sensitive to light for 6 weeks or moreafter treatment. Patients are advised to avoid direct sunlight andbright indoor light for at least 6 weeks. If patients must go outdoors,they need to wear protective clothing, including sunglasses. Othertemporary side effects of PDT are related to the treatment of specificareas and can include coughing, trouble swallowing, abdominal pain, andpainful breathing or shortness of breath. In December 1995, the U.S.Food and Drug Administration (FDA) approved a photosensitizing agentcalled porfimer sodium, or Photofrin®, to relieve symptoms of esophagealcancer that is causing an obstruction and for esophageal cancer thatcannot be satisfactorily treated with lasers alone. In January 1998, theFDA approved porfimer sodium for the treatment of early nonsmall celllung cancer in patients for whom the usual treatments for lung cancerare not appropriate. The National Cancer Institute and otherinstitutions are supporting clinical trials (research studies) toevaluate the use of photodynamic therapy for several types of cancer,including cancers of the bladder, brain, larynx, and oral cavity.

Laser therapy involves the use of high-intensity light to destroy cancercells. This technique is often used to relieve symptoms of cancer suchas bleeding or obstruction, especially when the cancer cannot be curedby other treatments. It may also be used to treat cancer by shrinking ordestroying tumors. The term “laser” stands for light amplification bystimulated emission of radiation. Ordinary light, such as that from alight bulb, has many wavelengths and spreads in all directions. Laserlight, on the other hand, has a specific wavelength and is focused in anarrow beam. This type of high-intensity light contains a lot of energy.Lasers are very powerful and may be used to cut through steel or toshape diamonds. Lasers also can be used for very precise surgical work,such as repairing a damaged retina in the eye or cutting through tissue(in place of a scalpel). Although there are several different kinds oflasers, only three kinds have gained wide use in medicine: Carbondioxide (CO2) laser—This type of laser can remove thin layers from theskin's surface without penetrating the deeper layers. This technique isparticularly useful in treating tumors that have not spread deep intothe skin and certain precancerous conditions. As an alternative totraditional scalpel surgery, the CO2 laser is also able to cut the skin.The laser is used in this way to remove skin cancers.Neodymium:yttrium-aluminum-garnet (Nd:YAG) laser—Light from this lasercan penetrate deeper into tissue than light from the other types oflasers, and it can cause blood to clot quickly. It can be carriedthrough optical fibers to less accessible parts of the body. This typeof laser is sometimes used to treat throat cancers. Argon laser—Thislaser can pass through only superficial layers of tissue and istherefore useful in dermatology and in eye surgery. It also is used withlight-sensitive dyes to treat tumors in a procedure known asphotodynamic therapy (PDT). Lasers have several advantages over standardsurgical tools, including: Lasers are more precise than scalpels. Tissuenear an incision is protected, since there is little contact withsurrounding skin or other tissue. The heat produced by lasers sterilizesthe surgery site, thus reducing the risk of infection. Less operatingtime may be needed because the precision of the laser allows for asmaller incision. Healing time is often shortened; since laser heatseals blood vessels, there is less bleeding, swelling, or scarring.Laser surgery may be less complicated. For example, with fiber optics,laser light can be directed to parts of the body without making a largeincision. More procedures may be done on an outpatient basis. Lasers canbe used in two ways to treat cancer: by shrinking or destroying a tumorwith heat, or by activating a chemical—known as a photosensitizingagent—that destroys cancer cells. In PDT, a photosensitizing agent isretained in cancer cells and can be stimulated by light to cause areaction that kills cancer cells. CO2 and Nd:YAG lasers are used toshrink or destroy tumors. They may be used with endoscopes, tubes thatallow physicians to see into certain areas of the body, such as thebladder. The light from some lasers can be transmitted through aflexible endoscope fitted with fiber optics. This allows physicians tosee and work in parts of the body that could not otherwise be reachedexcept by surgery and therefore allows very precise aiming of the laserbeam. Lasers also may be used with low-power microscopes, giving thedoctor a clear view of the site being treated. Used with otherinstruments, laser systems can produce a cutting area as small as 200microns in diameter—less than the width of a very fine thread. Lasersare used to treat many types of cancer. Laser surgery is a standardtreatment for certain stages of glottis (vocal cord), cervical, skin,lung, vaginal, vulvar, and penile cancers. In addition to its use todestroy the cancer, laser surgery is also used to help relieve symptomscaused by cancer (palliative care). For example, lasers may be used toshrink or destroy a tumor that is blocking a patient's trachea(windpipe), making it easier to breathe. It is also sometimes used forpalliation in colorectal and anal cancer. Laser-induced interstitialthermotherapy (LITT) is one of the most recent developments in lasertherapy. LITT uses the same idea as a cancer treatment calledhyperthermia; that heat may help shrink tumors by damaging cells ordepriving them of substances they need to live. In this treatment,lasers are directed to interstitial areas (areas between organs) in thebody. The laser light then raises the temperature of the tumor, whichdamages or destroys cancer cells.

The duration and/or dose of treatment with cancer therapies may varyaccording to the particular cancer agent or combination thereof. Anappropriate treatment time for a particular cancer therapeutic agentwill be appreciated by the skilled artisan. The invention contemplatesthe continued assessment of optimal treatment schedules for each cancertherapeutic agent, where the phenotype of the cancer of the subject asdetermined by the methods of the invention is a factor in determiningoptimal treatment doses and schedules.

In general, it is preferable to obtain a first sample from the subjectprior to beginning therapy and one or more samples during treatment. Insuch a use, a baseline of expression of cells from subjects with canceror cell hyperproliferative disorders prior to therapy is determined andthen changes in the baseline state of expression of cells from subjectswith cancer or cell hyperproliferative disorders is monitored during thecourse of therapy. Alternatively, two or more successive samplesobtained during treatment can be used without the need of apre-treatment baseline sample. In such a use, the first sample obtainedfrom the subject is used as a baseline for determining whether theexpression of cells from subjects with cancer or cell hyperproliferativedisorders is increasing or decreasing.

Another aspect of the present invention relates to a method ofpredicting the efficacy of a cancer therapy in a subject, comprisingobtaining a biological sample from the subject, and comparing theamount, structure, subcellular localization, and/or activity of at leastone marker selected from the group consisting of BAL1, BBAP, and/orBAL1-BBAP complex in a subject sample and the amount, structure,subcellular localization, and/or activity of the at least one marker ina control, wherein a significant difference in the amount, structure,subcellular localization, and/or activity of the at least one marker inthe sample and the amount, structure, subcellular localization, and/oractivity in the control is predictive of the outcome of treatment of thesubject with the cancer therapy. Methods described herein can use one ormore of various control samples, subjects, etc. For example, the controlcan be selected from the group consisting of a non-cancerous cell samplefrom the subject or member of the same species to which the subjectbelongs; anon-cancerous tissue that is the same tissue type as thecancerous tissue of the subject; and a non-cancerous tissue that is notthe same tissue type as the cancerous tissue of the subject.

In yet another aspect, a method for assessing the efficacy of an agentthat modulates the expression and/or activity of BAL1, BBAP, or aBAL-BBAP complex for enhancing the efficacy of a cancer therapy in asubject comprising detecting in a subject sample at a first point intime, the expression and/or activity of BAL1, BBAP, or a BAL-BBAPcomplex; repeating the previous step during at least one subsequentpoint in time after administration of the agent; and comparing theexpression and/or activity detected in the comparison, wherein asignificantly higher expression and/or activity of BAL1, BBAP, or aBAL-BBAP complex expression and/or activity in the first subject samplerelative to at least one subsequent subject sample, indicates that theagent enhances the efficacy of the cancer therapy in the subject and/orwherein a significantly decreased amount of an activity selected fromthe group consisting of a) increased binding to a BAL1 polypeptide orfragment thereof; b) increased binding of a BBAP polypeptide or fragmentthereof; c) increased formation of a BAL1-BBAP complex; d) inhibition oflocalization and/or binding of BAL1 and/or BBAP to DNA damage sites; e)inhibition of binding of BAL1 to poly(ADP-ribose) (PAR) chains; f)inhibition of BBAP monoubiquitylation of histones; g) inhibition ofBBAP-mediated methylation of histones; h) inhibition of localizationand/or binding to DNA damage sites of at least one polypeptide selectedfrom the group consisting of 53 BP1, RAP80, BRCA1, ATM, γH2AX, and MDC1;and i) inhibition DNA damage responses (DDR); in the first subjectsample relative to at least one subsequent subject sample, indicatesthat the test agent enhances the efficacy of the cancer therapy in thesubject.

Any means for the introduction of a polynucleotide into mammals, humanor non-human, or cells thereof may be adapted to the practice of thisinvention for the delivery of the various constructs of the inventioninto the intended recipient. In one embodiment of the invention, the DNAconstructs are delivered to cells by transfection, i.e., by delivery of“naked” DNA or in a complex with a colloidal dispersion system. Acolloidal system includes macromolecule complexes, nanocapsules,microspheres, beads, and lipid-based systems including oil-in-wateremulsions, micelles, mixed micelles, and liposomes. The preferredcolloidal system of this invention is a lipid-complexed orliposome-formulated DNA. In the former approach, prior to formulation ofDNA, e.g., with lipid, a plasmid containing a transgene bearing thedesired DNA constructs may first be experimentally optimized forexpression (e.g., inclusion of an intron in the 5′ untranslated regionand elimination of unnecessary sequences (Feigner, et al., Ann NY AcadSci 126-139, 1995). Formulation of DNA, e.g. with various lipid orliposome materials, may then be effected using known methods andmaterials and delivered to the recipient mammal. See, e.g., Canonico etal, Am J Respir Cell Mol Biol 10:24-29, 1994; Tsan et al, Am J Physiol268; Alton et al., Nat Genet. 5:135-142, 1993 and U.S. Pat. No.5,679,647 by Carson et al.

The targeting of liposomes can be classified based on anatomical andmechanistic factors. Anatomical classification is based on the level ofselectivity, for example, organ-specific, cell-specific, andorganelle-specific. Mechanistic targeting can be distinguished basedupon whether it is passive or active. Passive targeting utilizes thenatural tendency of liposomes to distribute to cells of thereticulo-endothelial system (RES) in organs, which contain sinusoidalcapillaries. Active targeting, on the other hand, involves alteration ofthe liposome by coupling the liposome to a specific ligand such as amonoclonal antibody, sugar, glycolipid, or protein, or by changing thecomposition or size of the liposome in order to achieve targeting toorgans and cell types other than the naturally occurring sites oflocalization.

The surface of the targeted delivery system may be modified in a varietyof ways. In the case of a liposomal targeted delivery system, lipidgroups can be incorporated into the lipid bilayer of the liposome inorder to maintain the targeting ligand in stable association with theliposomal bilayer. Various linking groups can be used for joining thelipid chains to the targeting ligand. Naked DNA or DNA associated with adelivery vehicle, e.g., liposomes, can be administered to several sitesin a subject (see below).

Nucleic acids can be delivered in any desired vector. These includeviral or non-viral vectors, including adenovirus vectors,adeno-associated virus vectors, retrovirus vectors, lentivirus vectors,and plasmid vectors. Exemplary types of viruses include HSV (herpessimplex virus), AAV (adeno associated virus), HIV (humanimmunodeficiency virus), BIV (bovine immunodeficiency virus), and MLV(murine leukemia virus). Nucleic acids can be administered in anydesired format that provides sufficiently efficient delivery levels,including in virus particles, in liposomes, in nanoparticles, andcomplexed to polymers.

The nucleic acids encoding a protein or nucleic acid of interest may bein a plasmid or viral vector, or other vector as is known in the art.Such vectors are well known and any can be selected for a particularapplication. In one embodiment of the invention, the gene deliveryvehicle comprises a promoter and a demethylase coding sequence.Preferred promoters are tissue-specific promoters and promoters whichare activated by cellular proliferation, such as the thymidine kinaseand thymidylate synthase promoters. Other preferred promoters includepromoters which are activatable by infection with a virus, such as theα- and β-interferon promoters, and promoters which are activatable by ahormone, such as estrogen. Other promoters which can be used include theMoloney virus LTR, the CMV promoter, and the mouse albumin promoter. Apromoter may be constitutive or inducible.

In another embodiment, naked polynucleotide molecules are used as genedelivery vehicles, as described in WO 90/11092 and U.S. Pat. No.5,580,859. Such gene delivery vehicles can be either growth factor DNAor RNA and, in certain embodiments, are linked to killed adenovirus.Curiel et al., Hum. Gene. Ther. 3:147-154, 1992. Other vehicles whichcan optionally be used include DNA-ligand (Wu et al., J. Biol. Chem.264:16985-16987, 1989), lipid-DNA combinations (Feigner et al., Proc.Natl. Acad. Sci. USA 84:7413 7417, 1989), liposomes (Wang et al., Proc.Natl. Acad. Sci. 84:7851-7855, 1987) and microprojectiles (Williams etal., Proc. Natl. Acad. Sci. 88:2726-2730, 1991).

A gene delivery vehicle can optionally comprise viral sequences such asa viral origin of replication or packaging signal. These viral sequencescan be selected from viruses such as astrovirus, coronavirus,orthomyxovirus, papovavirus, paramyxovirus, parvovirus, picornavirus,poxvirus, retrovirus, togavirus or adenovirus. In a preferredembodiment, the growth factor gene delivery vehicle is a recombinantretroviral vector. Recombinant retroviruses and various uses thereofhave been described in numerous references including, for example, Mannet al., Cell 33:153, 1983, Cane and Mulligan, Proc. Nat'l. Acad. Sci.USA 81:6349, 1984, Miller et al., Human Gene Therapy 1:5-14, 1990, U.S.Pat. Nos. 4,405,712, 4,861,719, and 4,980,289, and PCT Application Nos.WO 89/02,468, WO 89/05,349, and WO 90/02,806. Numerous retroviral genedelivery vehicles can be utilized in the present invention, includingfor example those described in EP 0,415,731; WO 90/07936; WO 94/03622;WO 93/25698; WO 93/25234; U.S. Pat. No. 5,219,740; WO 9311230; WO9310218; Vile and Hart, Cancer Res. 53:3860-3864, 1993; Vile and Hart,Cancer Res. 53:962-967, 1993; Ram et al., Cancer Res. 53:83-88, 1993;Takamiya et al., J. Neurosci. Res. 33:493-503, 1992; Baba et al., J.Neurosurg. 79:729-735, 1993 (U.S. Pat. No. 4,777,127, GB 2,200,651, EP0,345,242 and WO91/02805).

Other viral vector systems that can be used to deliver a polynucleotideof the invention have been derived from herpes virus, e.g., HerpesSimplex Virus (U.S. Pat. No. 5,631,236 by Woo et al., issued May 20,1997 and WO 00/08191 by Neurovex), vaccinia virus (Ridgeway (1988)Ridgeway, “Mammalian expression vectors,” In: Rodriguez R L, Denhardt DT, ed. Vectors: A survey of molecular cloning vectors and their uses.Stoneham: Butterworth; Baichwal and Sugden (1986) “Vectors for genetransfer derived from animal DNA viruses: Transient and stableexpression of transferred genes,” In: Kucherlapati R, ed. Gene transfer.New York: Plenum Press; Coupar et al. (1988) Gene, 68:1-10), and severalRNA viruses. Preferred viruses include an alphavirus, a poxivirus, anarena virus, a vaccinia virus, a polio virus, and the like. They offerseveral attractive features for various mammalian cells (Friedmann(1989) Science, 244:1275-1281; Ridgeway, 1988, supra; Baichwal andSugden, 1986, supra; Coupar et al., 1988; Horwich et al. (1990) J.Virol., 64:642-650).

In other embodiments, target DNA in the genome can be manipulated usingwell-known methods in the art. For example, the target DNA in the genomecan be manipulated by deletion, insertion, and/or mutation areretroviral insertion, artificial chromosome techniques, gene insertion,random insertion with tissue specific promoters, gene targeting,transposable elements and/or any other method for introducing foreignDNA or producing modified DNA/modified nuclear DNA. Other modificationtechniques include deleting DNA sequences from a genome and/or alteringnuclear DNA sequences. Nuclear DNA sequences, for example, may bealtered by site-directed mutagenesis.

In other embodiments, recombinant BAL1 and/or BBAP polypeptides, andfragments thereof, can be administered to subjects. In some embodiments,fusion proteins can be constructed and administered which have enhancedbiological properties (e.g., covalently bound BAL1-BBAP complexesdiscussed above). In addition, the BAL1 and/or BBAP polypeptides, andfragment thereof, can be modified according to well knownpharmacological methods in the art (e.g., pegylation, glycosylation,oligomerization, etc.) in order to further enhance desirable biologicalactivities, such as increased bioavailability and decreased proteolyticdegradation.

VI. Pharmaceutical Compositions

In another aspect, the present invention provides pharmaceuticallyacceptable compositions which comprise a therapeutically-effectiveamount of an agent that modulates (e.g., increases or decreases) BAL1and/or BBAP expression and/or activity, formulated together with one ormore pharmaceutically acceptable carriers (additives) and/or diluents.As described in detail below, the pharmaceutical compositions of thepresent invention may be specially formulated for administration insolid or liquid form, including those adapted for the following: (1)oral administration, for example, drenches (aqueous or non-aqueoussolutions or suspensions), tablets, boluses, powders, granules, pastes;(2) parenteral administration, for example, by subcutaneous,intramuscular or intravenous injection as, for example, a sterilesolution or suspension; (3) topical application, for example, as acream, ointment or spray applied to the skin; (4) intravaginally orintrarectally, for example, as a pessary, cream or foam; or (5) aerosol,for example, as an aqueous aerosol, liposomal preparation or solidparticles containing the compound.

The phrase “therapeutically-effective amount” as used herein means thatamount of an agent that modulates (e.g., enhances) BAL1 and/or BBAPexpression and/or activity, or expression and/or activity of thecomplex, or composition comprising an agent that modulates (e.g.,enhances) BAL1 and/or BBAP expression and/or activity, or expressionand/or activity of the complex, which is effective for producing somedesired therapeutic effect, e.g., weight loss, at a reasonablebenefit/risk ratio.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose agents, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically-acceptable carrier” as used herein means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting the subject chemical fromone organ, or portion of the body, to another organ, or portion of thebody. Each carrier must be “acceptable” in the sense of being compatiblewith the other ingredients of the formulation and not injurious to thesubject. Some examples of materials which can serve aspharmaceutically-acceptable carriers include: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21)other non-toxic compatible substances employed in pharmaceuticalformulations.

The term “pharmaceutically-acceptable salts” refers to the relativelynon-toxic, inorganic and organic acid addition salts of the agents thatmodulates (e.g., enhances) BAL1 and/or BBAP expression and/or activity,or expression and/or activity of the complex encompassed by theinvention. These salts can be prepared in situ during the finalisolation and purification of the respiration uncoupling agents, or byseparately reacting a purified respiration uncoupling agent in its freebase form with a suitable organic or inorganic acid, and isolating thesalt thus formed. Representative salts include the hydrobromide,hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate,valerate, oleate, palmitate, stearate, laurate, benzoate, lactate,phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate,napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonatesalts and the like (See, for example, Berge et al. (1977)“Pharmaceutical Salts”, J. Pharm. Sci. 66:1-19).

In other cases, the agents useful in the methods of the presentinvention may contain one or more acidic functional groups and, thus,are capable of forming pharmaceutically-acceptable salts withpharmaceutically-acceptable bases. The term “pharmaceutically-acceptablesalts” in these instances refers to the relatively non-toxic, inorganicand organic base addition salts of agents that modulates (e.g.,enhances) BAL1 and/or BBAP expression and/or activity, or expressionand/or activity of the complex. These salts can likewise be prepared insitu during the final isolation and purification of the respirationuncoupling agents, or by separately reacting the purified respirationuncoupling agent in its free acid form with a suitable base, such as thehydroxide, carbonate or bicarbonate of a pharmaceutically-acceptablemetal cation, with ammonia, or with a pharmaceutically-acceptableorganic primary, secondary or tertiary amine Representative alkali oralkaline earth salts include the lithium, sodium, potassium, calcium,magnesium, and aluminum salts and the like. Representative organicamines useful for the formation of base addition salts includeethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine,piperazine and the like (see, for example, Berge et al., supra).

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically-acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Formulations useful in the methods of the present invention includethose suitable for oral, nasal, topical (including buccal andsublingual), rectal, vaginal, aerosol and/or parenteral administration.The formulations may conveniently be presented in unit dosage form andmay be prepared by any methods well known in the art of pharmacy. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will vary depending upon thehost being treated, the particular mode of administration. The amount ofactive ingredient, which can be combined with a carrier material toproduce a single dosage form will generally be that amount of thecompound which produces a therapeutic effect. Generally, out of onehundred percent, this amount will range from about 1 percent to aboutninety-nine percent of active ingredient, preferably from about 5percent to about 70 percent, most preferably from about 10 percent toabout 30 percent.

Methods of preparing these formulations or compositions include the stepof bringing into association an agent that modulates (e.g., increases ordecreases) BAL1 and/or BBAP expression and/or activity, with the carrierand, optionally, one or more accessory ingredients. In general, theformulations are prepared by uniformly and intimately bringing intoassociation a respiration uncoupling agent with liquid carriers, orfinely divided solid carriers, or both, and then, if necessary, shapingthe product.

Formulations suitable for oral administration may be in the form ofcapsules, cachets, pills, tablets, lozenges (using a flavored basis,usually sucrose and acacia or tragacanth), powders, granules, or as asolution or a suspension in an aqueous or non-aqueous liquid, or as anoil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup,or as pastilles (using an inert base, such as gelatin and glycerin, orsucrose and acacia) and/or as mouth washes and the like, each containinga predetermined amount of a respiration uncoupling agent as an activeingredient. A compound may also be administered as a bolus, electuary orpaste.

In solid dosage forms for oral administration (capsules, tablets, pills,dragees, powders, granules and the like), the active ingredient is mixedwith one or more pharmaceutically-acceptable carriers, such as sodiumcitrate or dicalcium phosphate, and/or any of the following: (1) fillersor extenders, such as starches, lactose, sucrose, glucose, mannitol,and/or silicic acid; (2) binders, such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,sucrose and/or acacia; (3) humectants, such as glycerol; (4)disintegrating agents, such as agar-agar, calcium carbonate, potato ortapioca starch, alginic acid, certain silicates, and sodium carbonate;(5) solution retarding agents, such as paraffin; (6) absorptionaccelerators, such as quaternary ammonium compounds; (7) wetting agents,such as, for example, acetyl alcohol and glycerol monostearate; (8)absorbents, such as kaolin and bentonite clay; (9) lubricants, such atalc, calcium stearate, magnesium stearate, solid polyethylene glycols,sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents.In the case of capsules, tablets and pills, the pharmaceuticalcompositions may also comprise buffering agents. Solid compositions of asimilar type may also be employed as fillers in soft and hard-filledgelatin capsules using such excipients as lactose or milk sugars, aswell as high molecular weight polyethylene glycols and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered peptide orpeptidomimetic moistened with an inert liquid diluent.

Tablets, and other solid dosage forms, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions, which canbe dissolved in sterile water, or some other sterile injectable mediumimmediately before use. These compositions may also optionally containopacifying agents and may be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain portion ofthe gastrointestinal tract, optionally, in a delayed manner Examples ofembedding compositions, which can be used include polymeric substancesand waxes. The active ingredient can also be in micro-encapsulated form,if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups andelixirs. In addition to the active ingredient, the liquid dosage formsmay contain inert diluents commonly used in the art, such as, forexample, water or other solvents, solubilizing agents and emulsifiers,such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, oils (in particular, cottonseed, groundnut, corn, germ, olive,castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethyleneglycols and fatty acid esters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active agent may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Formulations for rectal or vaginal administration may be presented as asuppository, which may be prepared by mixing one or more respirationuncoupling agents with one or more suitable nonirritating excipients orcarriers comprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active agent.

Formulations which are suitable for vaginal administration also includepessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining such carriers as are known in the art to be appropriate.

Dosage forms for the topical or transdermal administration of an agentthat modulates (e.g., increases or decreases) BAL1 and/or BBAPexpression and/or activity include powders, sprays, ointments, pastes,creams, lotions, gels, solutions, patches and inhalants. The activecomponent may be mixed under sterile conditions with apharmaceutically-acceptable carrier, and with any preservatives,buffers, or propellants which may be required.

The ointments, pastes, creams and gels may contain, in addition to arespiration uncoupling agent, excipients, such as animal and vegetablefats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc and zincoxide, or mixtures thereof.

Powders and sprays can contain, in addition to an agent that modulates(e.g., increases or decreases) BAL1 and/or BBAP expression and/oractivity, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

The agent that modulates (e.g., increases or decreases) BAL1 and/or BBAPexpression and/or activity, can be alternatively administered byaerosol. This is accomplished by preparing an aqueous aerosol, liposomalpreparation or solid particles containing the compound. A nonaqueous(e.g., fluorocarbon propellant) suspension could be used. Sonicnebulizers are preferred because they minimize exposing the agent toshear, which can result in degradation of the compound.

Ordinarily, an aqueous aerosol is made by formulating an aqueoussolution or suspension of the agent together with conventionalpharmaceutically acceptable carriers and stabilizers. The carriers andstabilizers vary with the requirements of the particular compound, buttypically include nonionic surfactants (Tweens, Pluronics, orpolyethylene glycol), innocuous proteins like serum albumin, sorbitanesters, oleic acid, lecithin, amino acids such as glycine, buffers,salts, sugars or sugar alcohols. Aerosols generally are prepared fromisotonic solutions.

Transdermal patches have the added advantage of providing controlleddelivery of a respiration uncoupling agent to the body. Such dosageforms can be made by dissolving or dispersing the agent in the propermedium. Absorption enhancers can also be used to increase the flux ofthe peptidomimetic across the skin. The rate of such flux can becontrolled by either providing a rate controlling membrane or dispersingthe peptidomimetic in polymer matrix/gel.

Ophthalmic formulations, eye ointments, powders, solutions and the like,are also contemplated as being within the scope of this invention.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise one or more respiration uncoupling agents incombination with one or more pharmaceutically-acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containantioxidants, buffers, bacteriostats, solutes which render theformulation isotonic with the blood of the intended recipient orsuspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption such as aluminum monostearate andgelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolution,which, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsule matrices of anagent that modulates (e.g., increases or decreases) BAL1 and/or BBAPexpression and/or activity, in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions, which are compatible with body tissue.

When the respiration uncoupling agents of the present invention areadministered as pharmaceuticals, to humans and animals, they can begiven per se or as a pharmaceutical composition containing, for example,0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient incombination with a pharmaceutically acceptable carrier.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be determined by the methods of thepresent invention so as to obtain an amount of the active ingredient,which is effective to achieve the desired therapeutic response for aparticular subject, composition, and mode of administration, withoutbeing toxic to the subject.

The nucleic acid molecules of the invention can be inserted into vectorsand used as gene therapy vectors. Gene therapy vectors can be deliveredto a subject by, for example, intravenous injection, localadministration (see U.S. Pat. No. 5,328,470) or by stereotacticinjection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA91:3054 3057). The pharmaceutical preparation of the gene therapy vectorcan include the gene therapy vector in an acceptable diluent, or cancomprise a slow release matrix in which the gene delivery vehicle isimbedded. Alternatively, where the complete gene delivery vector can beproduced intact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system.

EXEMPLIFICATION

This invention is further illustrated by the following examples, whichshould not be construed as limiting.

Example 1: Materials and Methods for Examples 2-9

A. Protein Depletion by siRNA

All siRNA oligonucleotides targeting DNA damage response factors (Table2) were synthesized by Integrated DNA Technologies (Coralville, Iowa).Thereafter, the siRNAs and the control non-targeting siRNA (10 μM) weretransfected into Hela cells using X-tremeGENE siRNA transfection reagent(Roche, Basel, Switzerland) according to the manufacturer'sinstructions. Cells were cultured for 48 hr., lysed and subjected toNu-PAGE and Western blotting as described in Yan et al. (2009) Mol. Cell36:110-120. The efficiency of respective protein knockdown was assessedby immunoblotting with the following individual antibodies: BAL1(Takeyama et al. (2003) J. Biol. Chem. 278:21930-21937); rabbitpolyclonal antibody (Abeam Cambridge, Mass., #ab53796); PARP1 (mousemonoclonal antibody, BD Biosciences, Franklin Lakes, N.J., #51-6639GR);BBAP (mouse monoclonal antibody, Takeyama et al. (2003) J. Biol. Chem.278:21930-21937 and Yan et al. (2009) Mol. Cell 36:110-120); ATM (mousemonoclonal antibody, Santa Cruz Biotechnology, Santa Cruz, Calif.#23922); and MDC1 (rabbit polyclonal antibody, Bethyl Labs, Montgomery,Tex., #A300-051A).

TABLE 2(siRNA sequences are disclosed as SEQ ID NOs: 13-26, respectively, inorder of appearance) Location 3′ DNA Gene* RNA sequences (bp)overhanging BAL1 5′_GCCCACGCAUGGAUCCAAAGAAU_3′  1915-1937 CC siRNA#15′_GGAUUCUUUGGAUCCAUGCGUGGGCCU_3′  1939-1313 BAL15′_CCCAYACCAGYYCYGCAAYGYGG_3′  2448-2470 TA siRNA#25′_UACCACAUUGCAGAACUGGUAUGGGAC_3′  2472-2446 BAL15′_GGAAGUAGCUCUCCGCUUCCUU_3′   160-184 C siRNA#35′_CGAAGGAAGCUGGAGAGCUACUUCCAG_3′   186-158 PARP15′_CCAAAGGAAGGAACGCUAACAAU_3′  3758-3780 TT siRNA5′_AAAUUGUUAGCGUUCCUUCCUUUGGUC_3′  3782-3756 ATM5′_AGCUAUCAGAGAAGCUAAUAAAU_3′ 12710-12732 TA siRNA5′_UAAUUUAUUAGCUUCUCUGAUAGCUUC_3′ 12734-12708 MDC15′_CCACUAGGAGAAAGACAAAUAGG_3′  5350-5372 TC siRNA5′_GACCUAUUUGUCUUUCUCCUAGUGGCC_3′  5374-5348 RNF85′_CCCCUUGUACAUAUAUCUUUAGAG_3′  3662-3684 AG siRNA5′_CUCUCUAAAGAUAUAUGUACAAGGGUG_3′  3685-3650 *NCBI references accessnumber: BAL1, NM_031458.2; PARP1, NM_001618.3; ATM, N000051.3; MDC1,NM_014641.2; RNF8, NM_003958 **5′-UTR specific BAL1 siRNA

B. Generation of BAL1, BBAP and PARP1 Constructs

The specific oligonucleotides used to generate GFP-tagged BAL1, BBAP andPARP1 constructs are listed in Table 3. Human PARP1 cDNA was purchasedfrom OriGene Technologies (#SC119157, Rockville, Md.). cDNAs for humanBAL1, BBAP (NCBI Reference Sequence: NM_031458.2.) (Aguiar et al. (2000)Blood 96:4328-4334 and Takeyama et al. (2003) J. Biol. Chem.278:21930-21937) and PARP1 were inserted by PCR into pcDNA3.1/NT-GFPTOPO(Invitrogen, Carlsbad, Calif.) to generate the respective N-terminaltagged proteins, GFP-BAL1, GFP-BBAP, GFP-PARP1. The BAL1 cDNA was alsocloned into pcDNA3.1-CT-GFP-TOPO (Invitrogen) to generate the C-terminalGFP-tagged protein (BAL1-GFP) (Supplemental Information). The GFP-taggedBAL1 domain constructs, Macros-BBD and Macro1 (FIG. 2F), were generatedby introducing a stop codon (nt2454, C-A; nt1202, C-T) into the GFP-BAL1plasmid with appropriate DNA oligonucleotides and the QuikChangeSite-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara,Calif.). Macro2-BBD and Macro2A were generated with appropriate PCRproducts cloned into pcDNA3.1/NT-GFP-TOPO; Macro2 was constructed byintroducing a stop codon (nt1913, C-A) into Macro2-BBD. D126A and IE326,327AA (IE-AA) were produced with appropriate oligonucleotides (nt704,A-C; nt1303, nt1304, nt1307, AT, A-GC, C) and site-directed mutagenesis.The double mutation BAL1 construct (DM) was generated by introducing theIE326,327AA mutation into the D126A construct.

TABLE 3 (Oligo and primer sequences are disclosed as SEQ ID NOs: 27-46,respectively, in order of appearance) Location* Wild Vector namePair of Oligos or primers (bp) type (bp) GFP-BAL15′_atggacttttccatggtggccggag_3′  104-128 5′_ttaatcaacaggctgccaccttg_3′2563-2541 Macros- 5′_gtttcagcaagtcccataAcagttctgcaatgtggt_3′ 2336-2471C nt2454 BBD 5′_accacattgcagaactgTtatgggacttgctgaaac_3′ 2471-2336 GMacro1 5′_gggaagagtgagctgggacaaTaaaccaccccttctttc_3′ 1216-1255 C nt12025′_gaaagaaggggtggtttattgtcccagctcactcttccc_3′ 1255-1216 G Macro2-5′_ctagggaagagtgagctgggacaagaaacc_3′ 1213-1242 BBD5′_acccacattgcagaactgttatgggacttgctgaaac_3′ 2471-2336 G nt2454 Marco25′_ctaggaagagtgagctgggacaagaaacc_3′ 1213-12425′_ggatccatgcgtgggcctAttacatctcttccacgttg_3′ 1930-1893 C nt1913 Macro2Δ5′_tggcagacggcagatgtaattgtt_3′ 1309-13325′_ggatccatgcgtgggcctAttacatctcttccacgttg_3′ 1930-1893 C nt1913 D126A5′_cagtctggaaagatgCcctcaccacacatgctg_3′  689-721 A nt7075′_cagcatgtgtggtgagGcatctttccagactg_3′  721-689 T IE326,5′_gtccaggccacGCtgCatggcagacggcagatg_3′ 1291-1324 AT 327AAnt1303, nt1304, A nt1307 5′_catctgccgtctgccatGcaGCgtggccctggac_3′1324-1291 T, TA GFP-BBAP 5′_atggcctccacctgcgcccgccgtc_3′   90-1155′_ttactcaattcctttgctttcagctc_3′ 2312-2285 GFP-5′_atggcggagtcttcggataagctc_3′  172-295 PARP15′_ttaccacagggaggtcttaaaattg_3′ 3216-3192 Muatated bases in theoligonucleotides are capitalized and their positions indicated (rightcolumn) *NCBI references access number: BAL1, NM_031458.2; BBAP,NM_138287.3; PARP1, NM_001618.3.

C. Cell Culture and In Vivo Expression of GFP-Tagged Proteins

Hela and 293T human embryonic kidney cells (ATCC) were grown in DMEMcontaining 10% fetal bovine serum (Invitrogen). Hela or 293 cells weretransiently transfected with the indicated expression plasmids(GFP-tagged BAL1, BAL1 domains, BBAP or PARP1) for 24 hrs prior tofurther analysis.

In additional experiments, 5′-UTR specific (Table 3) BAL1- or controlsiRNA-treated Hela cells were transfected with GFP vector, GFP-BAL1 orGFP-BAL1-DM constructs; 24 hours later, these cells were cultured in thepresence or absence of Dox (50 ng/ml) for an additional 24 hrs andviability was analyzed thereafter with anti-Annexin V and PI.

D. Laser Microirradiation

Cells were initially seeded on coverslips and sensitized with 10 μM5-bromo-2′deoxyuridine (BrdU, Roche) in phenol red-free medium(Invitrogen) for 24 h at 37° C. In selected experiments, the PARPinhibitor PJ34 (51JM) (Sigma, St. Louis, Mo.) was added to the culturemedium for 1 hr prior to laser microirradiation. Laser microirradiationwas carried out on a Zeiss LSM51 0 NLO confocal microscope (Carl ZeissMicroImaging, LLC, Thornwood, N.Y.) equipped with a Coherent Chameleonpulse laser focused through a 40× LD C-Apochromat NA 0.9 water immersionobjective to yield a spot size of 0.5-1 μm. Cells were exposed to theTi-Sapphire pulse laser (740 nm) for ˜200 ms (fast scanning mode) at 30%laser power for 5-10 iterations. These settings generated a detectabledamage response restricted to the laser path without noticeablecytotoxicity to the cells.

To ensure that cells with GFP-tagged proteins were assayed, cells withmoderate levels of GFP were systematically chosen using identical Argonlaser (488 nm) settings. The association and dissociation kinetics ofGFP-tagged proteins at sites of laser microirradiation were monitored onthe same microscope by measuring GFP fluorescence over time in thedamaged region using the 488 nm argon laser. The images were detectedand stored using Carl Zeiss AIM software (Carl Zeiss MicroImaging).Variations in fluorescence intensity (I) were plotted as a function oftime (t) using the Zeiss AIM software. Data were normalized against thefluorescence intensity at the right time before microirradiation (I0).The deviations in GFP at each time point and condition were determinedby averaging values from >10 cells from a representative experiment.

E. Analysis of DNA Damage Sites of Laser Microirradiation by ConfocalMicroscopy

Hela cells were grown on coverslips and subjected to lasermicroirradiation as described above. Indirect immunofluorescence wasperformed as previously described in Yan et al. (2009) Mol. Cell36:110-120 with minor modifications. Primary antibodies included BAL1(Abcam, ab53796); PARP1 (rabbit monoclonal antibody, #9532, CellSignaling Technology, Danvers, Mass. or mouse monoclonal antibody, BDBiosciences, #51-6639); anti-PAR (Trevigen, Gaithersburg, Md., #4335);γH2AX (Millipore, Billerica, Mass., #05-636); BBAP (mouse monoclonalantibody, Yan et al. (2009) Mol. Cell 36:110-120) or rabbit polyclonalantibody, Bethyl, #A300-833A); FK2 (multi-ubiquitin monoclonal antibody,Enzo Life Sciences International, Plymouth Meeting, Pa., #SPA-205); ATM(mouse monoclonal antibody, Santa Cruz Biotechnology, #23922); MDC1(rabbit polyclonal antibody, Bethyl, # A300-051A); RAP80 (rabbitpolyclonal antibody, Bethyl, # A300-763A); BRCA1 (Millipore, #07-434);53BP1 (Santa Cruz Biotechnology, #22760); ATM-P-1981 (Millipore, clone10H11.E12); and RNF8 (rabbit polyclonal antibody, Abcam, #ab4183). Aftermultiple PBS washes, coverslips were incubated with FITC orCy5-conjugated secondary antibody raised against mouse or rabbit (AlexaFluor, Molecular Probes, Invitrogen) for 45 min and stained with PBScontaining DAPI (counterstain for DNA). Slides were mounted inVectashield mounting medium (Vector Labs, Burlingame, Calif.) afteradditional PBS washes. Confocal images were acquired on LSM-510 (CarlZeiss Microimaging) mounted on Zeiss-Axiovert 100M equipped withPlan-Neofluar 403/1.3 oil immersion objective as previously reported inYan et al. (2009) Mol. Cell 36:110-120. All images for a given conditionand immunostain over time were obtained with the same image acquisitionsettings.

F. Low-Dose Irradiation and Foci Formation

Gamma irradiation was performed as previously described in Yan et al.(2009) Mol. Cell 36:110-120 with minor modifications. In brief, Helacells grown on coverslips were treated with low-dose irradiation (100cGY) and analyzed at serial timepoints for repair foci by immunostainingwith the following antibodies: anti-BAL1 (Abcam, ab53796); anti-PAR(Trevigen, Gaithersburg, Md., #4335); anti-53BP1 (Santa CruzBiotechnology, #22760); and anti-γH2AX (Millipore, Billerica, Mass.,#05-636).

53BP1 and γH2AX repair foci were identified and counted with an Image Jmacro program. In brief, DAPI staining was utilized to create a nuclearmask. The numbers and intensity of repair foci within the nuclear maskwere captured by applying a 3p, rolling ball background reduction andmaximum entropy threshold algorithm to both Cy5 (γH2AX) and FITC (53BP1)channels.

G. Co-Immunoprecipitation Assays

Hela cells were incubated with PJ-34 or medium alone for 1 hour, treatedwith 50 ng/ml of doxorubicin for 10 min or left untreated; subsequently,cells were harvested and lysed in Triton buffer (TBS, 1% Triton X-1 00with protein inhibitors). Thereafter, cell lysates were incubated withanti-PARP1 (rabbit) antibody, anti-BAL1 (rabbit) antibody or controlrabbit lgG overnight at 4° C. Protein A Sepharose beads weresubsequently added to bind the lgG. After multiple washes in Tritonbuffer, antibody-associated proteins were released by boiling in proteinsample buffer, resolved by Nu-PAGE and immunoblotted with anti-PARP1(mouse), anti-PAR, or anti-BBAP (mouse) followed by donkey antimouse HRPor anti-SAL 1 antibody followed by protein A labeled HRP. In inputsamples, anti-actin antibody was used as a loading control.

H. Analysis of Cellular Proliferation and Apoptosis Following BAL1Depletion and Dox Treatment

Hela cells, which have high levels of endogenous BAL1, were firsttransfected with control scrambled or BAL1 siRNAs. Thereafter,siRNA-transfected or parental cells were seeded into 96-well plates at1×10⁴ cells/well, left untreated or treated with doxorubicin, 50 ng/ml,200 ng/ml or 400 ng/ml for 1-96 hrs and subsequently evaluated by MTSassay (CellTiter96® Aqueous Non-Radioactive Assay, Promega Corporation,Madison, Wis.). Assays were read at an absorbance of 490 nm using aSpectraMax 190 microplate reader (Molecular Devices, Sunnyvale, Calif.).All assays were performed in triplicate.

In additional studies, parental or BAL1- or control siRNA-treated Helacells were cultured in the presence or absence of Dox (50 or 200 ng/ml)for 24 hrs. Thereafter, the cells were detached with trypsin, washedwith PBS and incubated with anti-Annexin V antibody and PI in 100 μl ofbinding buffer (10 mM Hepes/NaOH, pH7.4, 140 mM NaCl, 2.5 mM CaCl) for15 minutes in the dark. Following the addition of 400 μl more bindingbuffer, the samples were analyzed by flow cytometry (Cytomics FC500,Beckman Coulter, Fullerton, Calif.).

I. Comet Assay

DNA damage was evaluated using the alkaline comet assay according to themanufacturer's instructions (Trevigen). PJ-34-treated andsiRNA-transfected cells were IR-treated at dosage of 2 Gys and recoveredin normal medium for indicated timepoints before processing. Harvestedcells (3×10³) were mixed with 0.8% low melting agarose and layered ontoagarose-coated slides. Slides were then submerged into lysis buffer 12.5M NaCl, 100 mM EDTA, 10 mM Tris (pH 10.0) and 1% Triton X-1001 overnightat 4° C. After lysis, slides were incubated for 30 mM in electrophoresisbuffer (200 mM NaOH and 1 mM EDTA, pH>13). After electrophoresis (25mins, 21 V, 300 mA), slides were washed with 2× water and 70% ethanoland then air-dried. Slides were stained with 2 μg/ml SYBR Green. AverageComet Tail Moment was scored (50-100 cells/slide) from the confocalimages documented on LSM-510 (Carl Zeiss Microimaging) as previouslyreported in Yan et al. (2009) Mol. Cell 36:110-120. Tail moment (=% DNAin tail*tail Length) was generated by using software TriTek CometScore™.

J. Vector Construction, Protein Purification, and Analysis

Oligonucleotides and primers used to generate recombinant GST-taggedBAL1 and FLAG-tagged PARP1 proteins are included in Tables 3 and 4.Using pFLAG-CMV2-BAL1 as template (Takeyama et al. (2003) J. Biol. Chem.278:21930-21937), BAL1 was PCR-amplified with primers containing Sal Iand Not I restriction sites (Table 4). The Sal I- and Not I-digestedBAL1 PCR product was then ligated into the vector, pGEX-4T-2 (GEHealthcare Bio-Sciences Corp., Piscataway, N.J.), to generate pGEX-BAL1.Using pGEXBAL1 as template, the double mutation BAL1 construct (DM) wasgenerated by introducing the IE326,327AA (IE-AA) and D126A mutationsinto the pGEX-BAL1 vector with the indicated oligonucleotides (Table 3)and the QuikChange Site Directed Mutagenesis Kit (Agilent Technologies).The pGEX-BAL1 recombinant proteins were generated and purified aspreviously described in Aguiar et al. (2005) J. Biol. Chem.280:33756-33765. Purified BAL1 and PARP1 recombinant proteins weresubjected to NuPAGE and analyzed by Coomassie Blue staining.

Human PARP1 cDNA was purchased from OriGene Technologies (#SC119157).Using the PARP1 cDNA as a template, FLAG-tagged PARP1 was PCR-amplifiedwith the indicated primers in Table 4. The resulting PCR product wasligated into pET101 following the manufacturer's instructions(Invitrogen). FLAG-tagged PARP1 was purified using the FLAGimmunoprecipitation kit according to the manufacturer's instructions(Sigma). Purified PARP1 recombinant proteins were size-fractionated andanalyzed by Coomassie Blue staining.

TABLE 4 (Oligo and primer sequences are disclosed as SEQ ID NOs: 47-52,respectively, in order of appearance) Vector Location namePair of Olicios or primers (bp) BBAP-his65′_caccatggcctcccacctgcgcccgccgtc_3′   90-1155′_ctcaattcctttggctttcagctc_3′ 2312-2285 GST-5′_acccgGTCGACatggacttttccatggtggccggag_3′  107-128 BAL1*5′_caccGCGGCCGCttaatcaacagggctgccacttg_3′ 2563-2541 FLAG-5′_caccATGGACTACAAGGATGACGATGACAAGatggcggagt  172-295 cttcggataagctc_3′PARP1** 5′_ttaccacagggaggtcttaaaattg_3′ 3216-3192 *The RE sites arecapitalzed. **The DNA sequence encoding FLAG tag is capitalized.

K. In Vitro PARP Activity Assay

In vitro poly(ADP-ribose) activity assays were carried out as previouslydescribed in Aguiar et al. (2005) J. Biol. Chem. 280:33756-33765 withminor modifications. Reactions including ˜200 ng of FLAG-tagged PARP1and 0, 0.5 or 1 mM of NAD+(Roche Applied Science, Indianapolis, Ind.)substrate were incubated at 25° C. for 30 min in assay buffer (0.1 ml)containing 50 mM Tris-HCl, pH 8.0, 4 mM MgCl₂, 0.2 mM dithiothreitol,and 200 ng of activated DNA (Sigma). In certain assays, the PARPinhibitor, PJ-34 (Sigma), was included at 1 mM final concentration.Reactions were stopped by the addition of 20% trichloroacetic acid.Precipitated proteins were rinsed once in 5% trichloroacetic acid,suspended in SDS loading buffer, and fractionated by NuPAGE(Invitrogen). After size fractionation, the proteins were subjected toimmunoblotting with anti-PARP1 antibody (mouse monoclonal antibody, BDBiosciences, #51-6639); and anti-PAR (Trevigen, #4335).

L. GST Pulldown

GST-BAL1 or -BAL1 mutant protein (1 μg) was immobilized onglutathione-Sepharose 48 beads and incubated with PARP assay samples (asdescribed above) with or without NAD in 1 ml of TBST buffer (20 mM Tris[pH 7.0], 200 mM NaCl, 1% NP-40, 1 mM dithiothreitol [DTT], and 0.5 mMEDTA) for 5 min at 4° C. on a rotating wheel. After 5 washes with TBSTbuffer, the samples were size-fractionated and immunoblotted withanti-PARP1 (BD Biosciences) and anti-PAR (Trevigen) antibodies.

M. In Vitro Ubiquitylation Assay

For vector construction, the previously described human BBAP cDNA (Ahelet al. (2009) Science 325:1240-1243) was utilized. Using the BBAP cDNAas a template, BBAP was PCR-amplified with the indicated primers inTable 4. The resulting PCR product was ligated into pET101 following themanufacturer's instructions (Invitrogen). His6-tagged BBAP was purifiedusing the Ni-NTA Spin Kit (Qiagen) according to the manufacturer'sinstructions. Purified BBAP-his6 recombinant protein wassize-fractionated, analyzed by Coomassie Blue staining and utilized inthe in vitro ubiquitylation assay which was performed as previouslydescribed in Ahel et al. (2009) Science 325:1240-1243. Using recombinantpET-BBAP-his6 wild type with minor modifications to the method, wildtype ubiquitin, lysine 48 (K48)-only ubiquitin and lysine 63 (K63)-onlyubiquitin were purchased from Boston Biochem (Cambridge, Mass. 02139).For immunoblotting of specific ubiquitin-chain, anti-ubiquitin-K48rabbit monoclonal antibody (05-1307) and anti-ubiquitin-K63 rabbitmonoclonal antibody (05-1308) were purchased from Millipore.

Example 2: BAL1 and BBAP Recruit to Laser Microirradiation Sites

Although BBAP protects cells exposed to DNA damaging agents (Yan et al.(2009) Mol. Cell 36:110-120), neither BBAP nor its partner protein,BAL1, have been directly associated with a DDR and the function of BAL1remains undefined. For these reasons, N-terminal GFP-tagged BAL1 andBBAP were expressed in Hela cells and their recruitment to sites of DNAdamage induced by laser microirradiation was evaluated. Both GFP-taggedBAL1 and BBAP localized to the laser-induced DNA breaks in less than 1min with maximum recruitment for ˜10 min and subsequent releasethereafter (FIGS. 1A-1B). Similar results were obtained when Hela cellswere laser microirradiated and immunostained for endogenous BAL1 (FIG.1C). The macro domain-containing protein rapidly localized to DNA damagesites with peak BAL1 recruitment within several minutes and dispersalwithin the nuclear compartment in less than 60 min (FIG. 1C). EndogenousBBAP was recruited to sites of laser-induced DNA breaks with similarkinetics (FIG. 1D).

In additional studies, Hela cells were subjected to an alternativesource of DNA damage—low-dose irradiation (100 cGy)—and immunostainedfor BAL1 (FIG. 1E). BAL1 foci were detectable in less than 1 min, weremost prominent at 4 min, and decreased in numbers and intensity by 30min (FIG. 1E). Taken together, these data directly implicate the macrodomain-containing BAL1 protein and its partner E3 ligase, BBAP, in theearly stages of a DDR.

Example 3: BAL1 Macro Domain 2 is Required for Recruitment to DNA DamageSites

To assess the role of the BAL1 tandem macro domains in localization toDNA damage sites, we generated a series of GFP-tagged BAL1 constructsthat encoded both macro domains (Macro 1 and 2), the respective singlemacro domains or mutated versions lacking critical residues in one orboth macro regions (FIG. 2). The individual GFP-tagged BAL1 constructswere transfected into 293T cells which were subsequently subjected tolaser microirradiation and analyzed by confocal microscopy (FIG. 2).

GFP-tagged BAL1 proteins containing both macro domains (BAL1 [1-854],Macros-BBD [1-708]), the complete macro domain 2 (Macro 2-BBD [296-708]or Macro 2 [296-528]) efficiently localized to sites of laser-inducedDNA breaks (FIG. 2). In contrast, GFP-tagged BAL1 proteins including atruncated macro domain 2 (Macro 24 [328-528]) or macro domain 1 alone(Macro 1 [1-291]) did not localize to DNA damage sites (FIG. 2).Consistent with these findings, targeted mutation of critical aminoacids in macro domain 2 (IE326,327AA [IE-AA]) markedly reduced BAL1recruitment, whereas a macro domain 1 mutation (D126A) had no effect(FIG. 2). Therefore, BAL1 recruitment to DNA damage sites depends uponits macro domains and macro domain 2 plays a non-redundant and essentialrole.

Example 4: BAL1/MAP Complex Co-Localizes with PARP1 and PAR andPhysically Associates with PAR-n-Proteins Following a DNA DamageResponse

The kinetics of BAL1 recruitment to DNA damage sites were similar tothose reported for PARP1 and its product, PAR (Ahel et al. (2009)Science 325:1240-1243 and Timinszky et al. (2009) Nat. Struct. Mol.Biol. 16:923-929). It was postulated that BAL1 interacted with PARP1and/or PAR at sites of DNA damage and analyzed BAL1, PARP1 and PARrecruitment in Hela cells following laser microirradiation (FIG. 3A).PARP1 co-localized with its product, PAR, and with BAL1 in laser-inducedDNA breaks (FIG. 3A). In addition, BBAP co-localized with PARP1 and PARin DNA damage sites (FIG. 3B). Hela cells were also subjected tolow-dose irradiation and early co-localization of BAL1 and PAR repairfoci was observed (FIG. 3C).

The interactions between these proteins and protein modifications weresubsequently assessed by immunoprecipitation. To define the basis ofPARP1 interactions in these studies, the drug, PJ-34, which inhibitsPARP1 generation of PAR but leaves PARP1 DNA binding intact, was used(Timinszky et al. (2009) Nat. Struct. Mol. Biol. 16:923-929). Hela cellswere untreated or treated with low-dose Dox (50 mg) for 10 min with orwithout PJ-34 pretreatment. Thereafter, whole cell lysates were preparedand PARP1, BAL1 (and control IgG) were immunoprecipitated andimmunoblotted for PARP1, PAR-n-proteins, BAL1 and BBAP (FIG. 3D). Inputwhole cell lysates were similarly analyzed (FIG. 3D, left panel).

As expected, input lysates from Dox-treated cells had a modest increasein PAR formation, which was abrogated by PJ-34 (FIG. 3D, left panel).PARP1 immune complexes from Dox-treated cells contained more abundantPAR-n-proteins in addition to increased BAL1 and BBAP and PJ-34inhibited these interactions (FIGS. 3D, 4A, and 4B). Following Doxtreatment, BAL1 immune complexes also included increased PAR-n-proteinsunless PARP activity was inhibited by PJ-34 (FIG. 3D). These dataindicate that BAL1 binds PAR-n-proteins, which are more abundantfollowing DNA damage and PARP1 activation.

To distinguish between BAL1 binding to PAR-modified proteins, includingPARP1, or PARP1 itself, in vitro pulldown assays were performed usingGST-tagged BAL1 proteins and FLAG-tagged PARP1 (FIGS. 4C-4D). It wasfirst demonstrated that FLAG-tagged PARP1 was functionally active invitro, catalyzing the synthesis of PARP1-associated PAR chains in thepresence of NAD+ (FIG. 4E). GST-tagged BAL1 selectively pulled downPAR-modified PARP1 in a macro domain-dependent manner, but did not bindunmodified PARP1 protein (FIGS. 4F-4G), confirming that BAL1specifically binds to PAR.

Example 5: Recruitment of BAL1/BBAP to DNA Damage Sites is DependentUpon PARP Activity

Given the interaction between BAL1 and PAR-n-proteins (FIGS. 3D and 4),it was next asked whether PARP1 activity was required for BAL1 and BBAPrecruitment to DNA damage sites. GFP-tagged PARP1, BAL1 or BBAP wereexpressed in Hela cells and the cells were treated with PJ-34 or vehiclealone prior to laser microirradiation. As expected, PJ-34 did not impairGFP-PARP1 binding to laser-induced DNA breaks (FIG. 3E). However, PJ-34pretreatment inhibited the recruitment of GFP-tagged BAL1 and BBAP toDNA damage sites (FIGS. 3E-3F).

Similar results were obtained when Hela cells were pre-incubated withPJ-34 or vehicle alone, laser microirradiated and immunostained forendogenous PARP1, PAR, BAL1 or BBAP (FIG. 5A). Although PJ-34 did notalter PARP1 recruitment to DNA damage sites, the compound eliminatedPARP-mediated PAR formation (FIG. 5A, top panel) and abrogated BAL1 andBBAP recruitment to laser-induced DNA breaks (FIG. 5A, middle and bottompanels). PJ-34 treatment also inhibited irradiation-induced PAR and BALfoci formation in Hela cells (FIGS. 6A-6B). Taken together, these dataindicate that PARP activity and PAR formation are required for BAL1 andBBAP localization to DNA damage sites.

To define the hierarchy of PARP1, BAL1 and BBAP interactions, eachprotein was individually depleted by siRNA and subsequent DNA damageresponse was assessed (FIGS. 5B-5C). PARP1 was a focus because over 85%of in vivo PARP activity is attributed to this family member (Yelamos etal. (2006) EMBO J. 25:4350-4360). PARP1 depletion (FIG. 5C, panels g-l)abrogated DNA-damaged induced PARP1 localization (panel h) andPARP-mediated PAR formation (panel k). In addition, PARP1 knockdowninhibited BAL1 and BBAP recruitment to laser-induced DNA breaks (FIG.5C, panels g and j, respectively). In BAL1 or BBAP-depleted cells,activated PARP1 and PAR were still recruited to DNA-damage sites,confirming that PARP1 functions upstream of BAL1 and BBAP (FIG. 5C,panels n, q; and t, w, respectively). In BAL1-depleted cells, BBAP didnot accumulate in laser-induced DNA breaks (FIG. 5C, panel p). Incontrast, BAL1 localized to DNA damage sites in BBAP-depleted cells(FIG. 5C, panel s), placing BAL1 upstream of BBAP. Therefore, BAL1 isrecruited to a DNA damage site via PARP1-mediated PAR formation and BBAPlocalizes to the site via its interaction with BALE

Example 6: BAL1 Limits the Cellular Response to DNA-Damaging Agents

The function of BAL1 in tumor (Hela) cell growth and (Dox)-inducedcytotoxicity was next assessed by depleting endogenous BAL1 via siRNAand treating the cells with Dox (50-200 ng). Although BAL1 RNAi reducedthe growth of untreated cells, the consequences of BAL1 depletion weremost striking in cells treated with low-dose Dox (50 ng) (FIG. 7A). Forexample, after 72 hr of treatment with low-dose Dox (50 ng), cellularproliferation was ˜80% lower in BAL1-depleted cells than in control RNAior parental cells (FIG. 5A, p<0.01). BAL1 depletion also increasedcellular apoptosis in both untreated and Dox-treated cells (FIG. 7B).These data indicate that the macro domain-containing BAL1 proteinenhances tumor cell survival and decreases Dox-induced cytotoxicity.

To directly evaluate the role of PARP-dependent BAL1 recruitment ontumor cell viability, we depleted BAL1 by siRNA and depleted with GFPvector only, GFP-BAL1 or GFP-BAL1DM (which lacks the PAR binding domain;see FIG. 2). In BAL1-depleted cells, BAL1 recruitment to PARP-1associated laser-induced DNA breaks was restored by GFP-BAL1 but not byGFP-BAL1DM (FIG. 7C; panels b vs. c, h vs. i). GFP-BAL1 repletion alsolimited the apoptosis of BAL1-depleted cells at baseline and followingDox treatment, unlike GFP-BAL1DM (FIG. 7D). These data directly andspecifically associate the early macro domain- and PAR-dependentrecruitment of BAL1 to DNA damage sites (FIG. 7C) with enhanced tumorcell survival (FIG. 7D).

In some embodiments, a skilled artisan can perform similar experimentsusing a variety of cancer therapies, including PARP inhibitors (e.g.,inhibitors of PARP-1). It is believed that BAL1 limits the cellularresponse to PARP inhibitors (e.g., inhibitors of PARP-1).

Example 7: Early Ubiquitin Chain Formation at DNA Damage Sites RequiresPARP1, BAL1 and BBAP

After demonstrating the functional significance of BAL1 recruitment toPARylated DNA damage sites (FIG. 7), it was assessed whether a potentiallink between PARP1 activation, localization of BAL1 partner E3 ligase,BBAP, and conjugated ubiquitin chain formation existed. Hela cells werepre-treated with PJ-34 or vehicle alone, laser microirradiated andimmunostained for PARP1, BBAP and newly formed ubiquitin chains (usingthe FK2 antibody which recognizes conjugated ubiquitin) (FIGS. 8A, 8B,and 9). FK2 immunostaining was readily apparent from the earliestanalyzed timepoint (5 min) through the last assessment (60 min) (FIGS.8A and 9). Although PJ-34 did not impair PARP1 recruitment to DNA damagesites, the PARP inhibitor abrogated BBAP localization and earlyubiquitin chain formation (FK2 immunostaining) (FIGS. 8A, 8B, 9A, and9B). BAL1 depletion similarly decreased BBAP recruitment and earlyubiquitin chain formation at laser-induced DNA breaks (FIGS. 8D-8E).Depletion of endogenous PARP1 or BBAP prior to laser microirradiationalso decreased early FK2 immunostaining. The effects of chemical PARPinhibition or BAL1 depletion on ubiquitin chain formation were moststriking in the first few minutes following laser microirradiation(FIGS. 8A, 8D, and 9). Taken together, these data indicate that earlyubiquitin chain formation at DNA damage sites is dependent upon PARP1activity, BAL/BBAP recruitment and the BBAP E3 ligase.

Example 8: PARP1 Activation and BAL1/MAP Recruitment to DNA Damage Sitesare Independent of ATM and MDC1

Given the recently described ATM/MDC1-dependent ubiquitylation at DNAdamage sites (Doil et al. (2009) Cell 136:435-446 and Mailand et al.(2007) Cell 131:887-900), the relationship between ATM and MDC1accumulation and PARP1 activation at laser-induced DNA breaks wasassessed. Hela cells were pre-incubated with PJ-34 or vehicle alone,laser microirradiated and immunostained for endogenous ATM, MDC1 andPARP1 (FIG. 10). The chemical PARP inhibitor did not impair ATM or MDC1recruitment to DNA damage sites (FIG. 10A). In complementaryexperiments, depletion of ATM or MDC1 by siRNA (FIG. 10B) had no effecton PARP1 or BAL1 accumulation at laser-induced DNA breaks (FIG. 10C).Therefore, PARP1 activation and BAL1 recruitment are independent of ATMand MDC1 suggesting that there are two separately regulated pathways ofDNA damage-induced ubiquitylation. Consistent with this hypothesis,neither chemical PARP inhibition nor BAL1 depletion altered the delayedkinetics of accumulation of the ATM/MDC-1 dependent E3 ligase, RNF8, atDNA damage sites (FIGS. 8C and 9C). RNF8 was primarily detectable 40-60min following laser microirradiation (FIGS. 8C, 8F, and 9C), in contrastto BBAP which was seen from the earliest evaluated timepoint, 5 min,through 20-40 min (FIGS. 8B, 8E, and 9B).

Example 9: Functional Analyses of PARP1/BAL1/BBAP- andMDC1/RNF8-Associated DDRs

The functional consequences of the respective PARP1/PAR-dependentBAL1/BBAP and ATM/MDC1/RNF8 DDR pathways were compared using cometassays, which measure unrepaired DNA damage in individual cells with gelelectrophoresis (Olive and Banath (2006) Nat. Protocol. 1:23-29). Ininitial studies, Hela cells were pre-treated with PJ-34 or vehiclealone, subjected to low-dose irradiation (200 cGy) and analyzed by cometassay 15 min, 60 min or 24 hrs thereafter (FIG. 11A). In control cells,the intensity of the comet tails, which reflect unrepaired DNA damage,was modestly increased at 15 min, decreased at 60 min and then nearedbaseline by 24 hrs (FIG. 11A, left panel). In PJ-34 treated cells, comettail intensity was markedly increased at 15 min and persistentlyelevated through 24 hrs (FIG. 11A, right panel). BAL1- and BBAP-depletedcells had similarly increased comet tail intensity 15 min through 24 hrfollowing irradiation (FIGS. 11B and 11D, left and middle panels).Therefore, chemical PARP inhibition and BAL1 or BBAP depletion allmarkedly increase unrepaired DNA damage at early and later timepoints(FIGS. 11A, 11B, and 11D).

The apparent independence of PARP1/BAL1/BBAP and ATM/MDC1/RNF8recruitment and ubiquitylation at DNA damage sites (FIGS. 8-10) prompteda comparison of these pathways using the comet assay. In contrast toBAL1-depleted cells, MDC1-knockdown cells had a more delayed pattern ofunrepaired DNA damage (comet tail intensity) following irradiation (FIG.11B). When BAL1 and MDC1 were both depleted prior to irradiation, comettail intensity was greater than that of either single knockdown at 15min through 24 hr (FIG. 11B, right panel).

DNA damage was also more delayed in RNF8-depleted cells than inBBAP-knockdown cells (FIGS. 11C-11D). When both E3 ligases, BBAP andRNF8, were depleted prior to irradiation, comet tail intensity wasgreater than that of either single knockdown from early through latertimepoints (FIG. 11D). Taken together, these data indicate thatPARP1-dependent BAL1/BBAP-mediated DNA damage repair is functionallydistinct and non-redundant to that of ATM/MDC1/RNF8.

Example 10: Early 53BP1 Recruitment to DNA Damage Sites Requires PARP1,BAL1 and BBAP

It was previously found that depletion of the BBAP E3 ligase delayed theaccumulation of the checkpoint mediator, 53BP1, in repair foci ((Yan etal. (2009) Mol. Cell 36:110-120). Given the dependence of BBAP on PARP1activation and BAL1 recruitment, the consequences of chemical PARP1inhibition (PJ-34 treatment) on 53BP1 accumulation at DNA damage siteswere assessed (FIG. 12A). In PJ-34 treated cells, 53BP1 localization tolaser-induced DNA breaks was significantly delayed (FIG. 12A). Similarresults were obtained when endogenous PARP1, BAL1 or BBAP were depletedby siRNA prior to laser microirradiation (FIG. 12B; images obtained ˜20min following microirradiation). These data directly implicate PARP1activation and BAL1/BBAP recruitment in the early localization of 53BP1to DNA damage sites.

To further characterize potentially separate PARP1- andATM/MDC1/RNF8/H2AX-dependent pathways of 53BP1 accumulation, Hela cellswere pre-treated with PJ34 or vehicle alone, subjected to low-doseirradiation and analyzed for 53BP1 and H2AX foci at 0-60 min thereafter.At the earliest timepoints following irradiation (<30 min), there weresignificantly more 53BP1 foci in control cells than in PJ34-treatedcells (p=0.004; FIGS. 12C-12D). In contrast, H2AX foci formation wasmore delayed than that of 53BP1 and unaffected by treatment with PJ-34(p=NS; FIGS. 12C-12D). After irradiation, 53BP1 foci formation was alsomore rapid in control cells than in BAL1-depleted cells (p=0.001; FIGS.12E-12F). However, H2AX foci formation was more delayed and similar incontrol and BAL knockdown cells (p=NS; FIGS. 12E-12F). These data definethe initial PARP1- and BAL1-dependent, H2AX-independent recruitment of53BP1 to DNA damage sites.

Example 11: Early RAP80 and BRCA1 Localization to DNA Damage SitesRequires PARP1, BAL1 and BBAP

BRCA1 accumulates at DNA damage sites via the adaptor protein, RAP80,and its ubiquitin-interacting motifs. For these reasons, we alsoanalyzed the kinetics of BRCA1 and RAP80 accumulation at laser-inducedDNA breaks following chemical PARP inhibition. In PJ-34 treated cells,RAP80 and BRCA1 recruitment to DNA damage sites were markedly delayed(FIGS. 13A-13B).

RAP80 and BRCA1 localization to DNA damage sites were also delayed inBAL1-depleted cells (FIGS. 14A-14D). Furthermore, PARP1, BAL1 or BBAPknockdown all reduced early RAP80 and BRCA localization to laser-inducedDNA breaks (FIG. 13C; PARP1, panels h and k; BAL1, panels n and q; andBBAP, panels t and w, respectively). Consistent with these findings,BBAP ubiquitylated the RAP80 residue, K63 (Lok et al. (2012) Nuc. AcidsRes. 40:196-205), in addition to K48 (FIG. 15).

Taken together, these data indicate that PARP1-, BAL1 and BBAP-dependentubiquitylation provides initial access to the downstream mediators,RAP80 and BRCA1, at DNA damage sites (FIG. 13D). These studies defineseparate temporally and functionally distinct mechanisms of DNA-damageinduced ubiquitylation and recruitment of 53BP1 and BRCA1—an earlyPARP1, BAL1 and BBAP-dependent pathway and a laterphosphorylation-dependent, ATM/MDC1/RNF8-associated route (FIG. 13D).

More generally, a direct link between the initial rapid and short-livedPARylation at DNA damage sites, PAR-dependent recruitment of the BAL1macro domain-containing protein and its partner E3 ligase, BBAP, andBBAP-mediated ubiquitylation and localization of the checkpointmediators, 53BP1 and BRCA1 has been defined herein. The PARP1-dependentlocalization of BAL1 and BBAP functionally limits early and delayed DNAdamage and enhances cellular viability independent of ATM/MDC1/RNF8.These data firmly establish BAL and BBAP as bona fide DDR pathwaymembers and provide new insights into PARP-mediated DNA repair.

The kinetics of BAL1 and BBAP recruitment to laser-induced breaksreflect their early PAR-dependent localization at these sites. Thesefindings provide a mechanistic basis for the previously described delayin 53BP1 foci formation in BBAP-depleted cells (Yan et al. (2009) Mol.Cell 36:110-120). In the earlier studies, BBAP knockdown selectivelydecreased 53BP1 recruitment at early timepoints following low-dosedoxorubicin (50 ng) or γ-irradiation (100 cGy)-induced DNA damage;however, at later timepoints, the numbers of 53BP1 repair foci weresimilar in BBAP-depleted cells and controls (Yan et al. (2009) Mol. Cell36:110-120). The findings likely reflect selective impairment of earlyPAR-dependent, BAL1/BBAP-mediated 53BP1 recruitment with intact delayedATM/MDC1/RNF8-dependent events (FIG. 13D).

The present invention also defines an early wave of ubiquitylation atDNA damage sites that is dependent upon PARP1 activation, BAL1/BBAPrecruitment and BBAP E3 ligase activity and independent of ATM/MDC1 andRNF8. PARP1-dependent, BAL1/BBAP-mediated ubiquitylation promotes therapid and specific recruitment of 53BP1, RAP80 and BRCA1 to DNA damagesites. BBAP ubiquitylates histone H4 lysine 91 and increases theaccessibility of H4K20 to methylation and 53BP1 recruitment via itstandem tudor domain (Yan et al. (2009) Mol. Cell 36:110-120).BBAP-mediated ubiquitylation likely fosters RAP80/BRCA1 localization viaRAP80 UIMs. Given the time course and kinetics of PARP1 activation andATM/MDC1 phosphorylation, these studies define separate andcomplementary pathways of BBAP- and RNF8/RNF168-mediated ubiquitylationand recruitment of 53BP1 and BRCA1 to DNA damage sites. Functionalanalyses confirm the non-redundant role of PARP1-dependent BAL1/BBAPrecruitment and ubiquitylation on DDR (FIGS. 7 and 11).

The roles of BAL1 and BBAP in PARP1-dependent DNA damage repair haveadditional clinical implications. The BAL1 macro protein and its partnerE3 ligase were originally identified in a screen for genes that wereoverexpressed in treatment-resistant lymphomas. The results presentedherein provide a mechanistic basis for the earlier observations andsuggest that targeted inhibition of BAL1 and/or BBAP may increase theefficacy of chemotherapy (doxorubicin) or radiation treatment. It hasbeen determined herein that a direct link exists between PARP1activation and BRCA1 recruitment and BAL1 macro protein and BBAP E3ligase are implicated in these processes.

Example 12: BAL1 and BBAP Form a Complex Via Specific InteractionDomains

In order to determine the protein domains necessary for BAL1 and BBAP tointeract, BBAP deletion constructs and BAL1 deletion constructions weresystematically generated. FIG. 16 shows a schematic view of several BBAPdeletion constructs and FIG. 17 shows a schematic view of BBAP's BAL1binding domain based on co-immunoprecipitation experiments. Similarco-immunoprecipitation experiments were conducted in order to map BBAP'shomodimerization domain (FIG. 18), as well as the BBAP binding domain ofBAL1 (Example 4). A schematic diagram summarizing and annotating thedomain structures of BAL1 and BBAP, as well as the BBAP- andBAL1-interacting domains of the respective proteins, is shown in FIG.19.

Example 13: Double Knockdown of BAL1 and BBAP Increases Induction ofApoptosis in Cancer Cells and Increases the Sensitivity of Cancer Cellsto Chemotherapeutic Agents

Double knockdown of BAL1 and BBAP using siRNA-mediated depletiontechniques described in Example 2 were performed to determine whethersimultaneously reducing the expression of both BAL1 and BBAP increasesthe induction of apoptotic cells and sensitivity of cells to doxorubicintreatment of the HeLa cell line. FIG. 20 demonstrates that doubleknockdown of BAL1 and BBAP increases induction of apoptosis in cancercells and increases the sensitivity of cancer cells to chemotherapeuticagents.

INCORPORATION BY REFERENCE

The contents of all references, patent applications, patents, andpublished patent applications, as well as the Figures and the SequenceListing, cited throughout this application are hereby incorporated byreference.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1-51. (canceled)
 52. An isolated nucleic acid molecule selected from thegroup consisting of: a) an isolated nucleic acid molecule which encodesat least one BBAP binding domain of a BAL1 protein and which does notencode full-length BAL1; b) an isolated nucleic acid molecule whichencodes at least one BBAP binding domain of a BAL1 protein and whichdoes not encode one or more functional domain(s) of a BAL1 proteinselected from the group consisting of Macro1, Macro 2, and PARP domains;c) an isolated nucleic acid molecule which encodes a polypeptidecomprising an amino acid sequence having at least 70% identity to theamino acid sequence of 453-702 of SEQ ID NO:2 and which does not encodefull-length BAL1 and/or which does not encode one or more functionaldomain(s) of a BAL1 protein selected from the group consisting ofMacro1, Macro 2, and PARP domains; d) an isolated nucleic acid moleculewhich encodes a polypeptide consisting essentially of an amino acidsequence having at least 70% identity to the amino acid sequence of453-702 of SEQ ID NO:2 and which does not encode full-length BAL1 and/orwhich does not encode one or more functional domain(s) of a BAL1 proteinselected from the group consisting of Macro1, Macro 2, and PARP domains;e) an isolated nucleic acid molecule which encodes at least one BAL1binding domain of a BBAP protein and which does not encode full-lengthBBAP; f) an isolated nucleic acid molecule which encodes at least oneBAL1 binding domain of a BBAP protein and which does not encode one ormore functional domain(s) of a BBAP protein selected from the groupconsisting of BBAP dimerization and RING domains; g) an isolated nucleicacid molecule which encodes a polypeptide comprising an amino acidsequence having at least 70% identity to the amino acid sequence of423-617 of SEQ ID NO:10 and which does not encode full-length BBAPand/or which does not encode one or more functional domain(s) of a BBAPprotein selected from the group consisting of BBAP dimerization and RINGdomains; and h) an isolated nucleic acid molecule which encodes apolypeptide consisting essentially of an amino acid sequence having atleast 70% identity to the amino acid sequence of 423-617 of SEQ ID NO:10and which does not encode full-length BBAP and/or which does not encodeone or more functional domain(s) of a BBAP protein selected from thegroup consisting of BBAP dimerization and RING domains.
 53. An isolatednucleic acid molecule comprising a nucleotide sequence which iscomplementary to the nucleic acid sequence of claim
 52. 54. The isolatednucleic acid molecule of claim 52, further comprising a nucleic acidsequence encoding a heterologous polypeptide.
 55. The isolated nucleicacid molecule of claim 54, wherein the heterologous polypeptide isselected from the group consisting of a signal peptide, a peptide tag, adimerization domain, an oligomerization domain, an antibody, or anantibody fragment.
 56. A vector comprising the nucleic acid molecule ofclaim
 52. 57. The vector of claim 56, which is an expression vector. 58.A host cell transfected with the expression vector of claim
 56. 59. Amethod of producing a polypeptide comprising culturing the host cell ofclaim 58 in an appropriate culture medium to thereby produce thepolypeptide.
 60. The method of claim 59, wherein the host cell isselected from the group consisting of a bacterial cell, a eukaryoticcell, and a cell genetically engineered to express a selectable marker.61. The method of claim 59, further comprising the step of isolating thepolypeptide from the medium or host cell.
 62. An isolated polypeptideselected from the group consisting of: a) an isolated polypeptidefragment of a BAL1 protein comprising at least one BBAP binding domainand is not full-length BAL1; b) an isolated polypeptide fragment of aBAL1 protein comprising at least one BBAP binding domain and which lacksone or more functional domain(s) selected from the group consisting ofMacro1, Macro 2, and PARP domains; c) an isolated polypeptide comprisingan amino acid sequence that is at least 70% identical to the amino acidsequence of residues 453-702 of SEQ ID NO:2 and is not full-length BAL1and/or which lacks one or more functional domain(s) of a BAL1 proteinselected from the group consisting of Macro1, Macro 2, and PARP domains;d) an isolated polypeptide consisting essentially of an amino acidsequence that is at least 70% identical to the amino acid sequencecomprising residues 453-702 of SEQ ID NO:2 and is not full-length BAL1and/or which lacks one or more functional domain(s) of a BAL1 proteinselected from the group consisting of Macro1, Macro 2, and PARP domains;e) an isolated polypeptide fragment of a BBAP protein which is encodedby a nucleic acid molecule comprising a nucleotide sequence encoding atleast one BBAP binding domain and does not encode full-length BAL1; f)an isolated polypeptide fragment of a BAL1 protein which is encoded by anucleic acid molecule comprising a nucleotide sequence encoding at leastone BBAP binding domain and which does not encode one or more functionaldomain(s) selected from the group consisting of Macro1, Macro 2, andPARP domains; g) an isolated polypeptide which is encoded by a nucleicacid molecule comprising a nucleotide sequence encoding an amino acidsequence that is at least 70% identical to the amino acid sequence ofresidues 453-702 of SEQ ID NO:2 and which does not encode full-lengthBAL1 and/or which does not encode one or more functional domain(s) of aBAL1 protein selected from the group consisting of Macro1, Macro 2, andPARP domains; h) an isolated polypeptide which is encoded by a nucleicacid molecule consisting essentially of a nucleotide sequence encodingan amino acid sequence that is at least 70% identical to the amino acidsequence of residues 453-702 of SEQ ID NO:2 and does not encodefull-length BAL1 and/or does not encode one or more functional domain(s)of a BAL1 protein selected from the group consisting of Macro1, Macro 2,and PARP domains; i) an isolated polypeptide fragment of a BBAP proteincomprising at least one BAL1 binding domain and is not full-length BBAP;j) an isolated polypeptide fragment of a BBAP protein comprising atleast one BAL1 binding domain and which lacks one or more functionaldomain(s) selected from the group consisting of BBAP dimerization andRING domains; k) an isolated polypeptide comprising an amino acidsequence that is at least 70% identical to the amino acid sequence ofresidues 423-617 of SEQ ID NO:10 and is not full-length BBAP and/orwhich lacks one or more functional domain(s) of a BBAP protein selectedfrom the group consisting of BBAP dimerization and RING domains; l) anisolated polypeptide consisting essentially of an amino acid sequencethat is at least 70% identical to the amino acid sequence comprisingresidues 423-617 of SEQ ID NO:10 and is not full-length BBAP and/orwhich lacks one or more functional domain(s) of a BBAP protein selectedfrom the group consisting of BBAP dimerization and RING domains; m) anisolated polypeptide fragment of a BBAP protein which is encoded by anucleic acid molecule comprising a nucleotide sequence encoding at leastone BAL1 binding domain and does not encode full-length BBAP; n) anisolated polypeptide fragment of a BBAP protein which is encoded by anucleic acid molecule comprising a nucleotide sequence encoding at leastone BAL1 binding domain and which does not encode one or more functionaldomain(s) selected from the group consisting of BBAP dimerization andRING domains; o) an isolated polypeptide which is encoded by a nucleicacid molecule comprising a nucleotide sequence encoding an amino acidsequence that is at least 70% identical to the amino acid sequence ofresidues 423-617 of SEQ ID NO:10 and which does not encode full-lengthBBAP and/or which does not encode one or more functional domain(s) of aBBAP protein selected from the group consisting of BBAP dimerization andRING domains; and p) an isolated polypeptide which is encoded by anucleic acid molecule consisting essentially of a nucleotide sequenceencoding an amino acid sequence that is at least 70% identical to theamino acid sequence of residues 423-617 of SEQ ID NO:10 and does notencode full-length BBAP and/or does not encode one or more functionaldomain(s) of a BBAP protein selected from the group consisting of BBAPdimerization and RING domains.
 63. The isolated polypeptide of claim 62,wherein the isolated polypeptide maintains the ability to promote one ormore biological activities selected from the group consisting of: a)binding to a BAL1 polypeptide or fragment thereof; b) binding to a BBAPpolypeptide or fragment thereof; c) forming a BAL1-BBAP complex; d)inhibiting localization and/or binding of BAL1 and/or BBAP to DNA damagesites; e) inhibiting binding of BAL1 to poly(ADP-ribose) (PAR) chains;f) inhibiting BBAP monoubiquitylation of histones; g) inhibitingBBAP-mediated methylation of histones; h) inhibiting localization and/orbinding to DNA damage sites of at least one polypeptide selected fromthe group consisting of 53 BP1, RAP80, BRCA1, ATM, γH2AX, and MDC1; andi) inhibiting DNA damage responses (DDR).
 64. The isolated polypeptideof claim 62, wherein the isolated polypeptide contains one or moreconservative amino acid substitutions.
 65. The isolated polypeptide ofclaim 62, further comprising a heterologous polypeptide.
 66. Acomposition comprising the isolated polypeptide of claim 62 and apharmaceutically acceptable agent selected from the group consisting ofexcipients, diluents, and carriers.
 67. The isolated polypeptide ofclaim 62, wherein the polypeptide is immobilized on an object selectedfrom the group consisting of a cell, a metal, a resin, a polymer, aceramic, a glass, a microelectrode, a graphitic particle, a bead, a gel,a plate, an array, and a capillary tube. 68-92. (canceled)